CN115429781A - Application of sodium houttuyfonate in preparing medicine for preventing and treating stress cardiomyopathy - Google Patents

Abstract

The invention discloses an application of sodium houttuyfonate in preparing a medicament for preventing and treating stress cardiomyopathy. The invention adopts isoproterenol to prepare a mouse model of the stress cardiomyopathy, adopts a C57BL/6 mouse to carry out experiments, uses sodium houttuyfonate to treat and observe the improvement effect on the cardiac function and the cardiac fibrosis in the isoproterenol-induced mouse model, and determines the influence of the sodium houttuyfonate on the proliferation capacity of the cardiac fibroblasts. The experiment of the invention proves that the sodium houttuyfonate has the prevention and treatment effect on the stress cardiomyopathy and the heart protection effect; can reduce the cardiac hypofunction caused by the stress cardiomyopathy; sodium houttuyfonate can inhibit myocardial fibrosis, inhibit proliferation of myocardial fibroblast, and inhibit expression of fibrosis marker protein. The sodium houttuyfonate has good clinical application prospect in the aspect of products for preventing and treating heart hypofunction, myocardial fibrosis and the like caused by the stress cardiomyopathy.

Description

Application of Houttuyfonate Sodium in Preparation of Drugs for Prevention and Treatment of Stress Cardiomyopathy

technical field

The invention relates to the technical field of biomedicine, in particular to the application of houttuyfonate sodium in the preparation of drugs for preventing and treating stress cardiomyopathy.

Background technique

Stress-induced "Takotsubo" cardiomyopathy is a recently described acute myocardial infarction-like cardiovascular syndrome characterized by reversible left ventricular dysfunction with apical ballooning in patients without coronary artery disease, affecting cardiac direction. The ability of other parts of the body to pump blood, and often leads to heart failure. Usually associated with emotional or physical stressful events. Prolonged stress leads to cardiac remodeling, including cardiac hypertrophy, inflammation, and fibrosis, ultimately leading to left ventricular dysfunction and even death. Fibrosis (scarring) is an abnormal wound-healing response to cardiac injury characterized by excessive accumulation of extracellular matrix components, mainly composed of collagen. Cardiac fibrosis is a potential target for innovative treatments in heart failure due to evidence from preclinical and clinical studies that cardiac fibrosis is reversible, but currently available treatments are refractory to controlling myocardial fibrosis.

Houttuynia cordata is a traditional herb and vegetable found in Asian countries that has anti-oxidative stress and anti-inflammatory properties. Houttuyniatin is an extract of Houttuynia cordata, which has shown remarkable medicinal value in animal and cell models of many diseases, such as lipopolysaccharide (LPS)-induced acute lung injury (ALI), smoking-induced chronic obstructive chronic pulmonary disease (COPD), LPS-induced inflammation of bovine endometrial epithelial cells, abdominal aortic cerclage-induced ventricular hypertrophy, and myocardial infarction. Studies have shown that houttuyfonate sodium is rapidly distributed to different tissues after intravenous injection, with the highest concentration in lung tissue, followed by heart tissue, indicating that the heart is also one of its main target organs. However, whether it protects against fibrosis in stress cardiomyopathy remains unclear.

Contents of the invention

The object of the present invention is to provide the application of houttuyfonate sodium in the preparation of medicines for preventing and treating stress cardiomyopathy.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows: the application of Houttuyfonate Sodium in the preparation of drugs for the prevention and treatment of stress cardiomyopathy.

The invention also provides the application of houttuyfonate sodium in the preparation of medicines for preventing and treating myocardial fibrosis.

In the above technical solution, the drug inhibits the proliferation of cardiac fibroblasts induced by isoproterenol, and inhibits the expression of Col1a, a-SMA, MMP2 and TIMP2 in cardiac fibroblasts induced by transforming growth factor β.

In the above technical solution, the medicine includes an effective dose of houttuyfonate sodium and a pharmaceutically acceptable carrier.

In the above technical solution, the dosage of houttuyfonate sodium is 100 mg/kg·day.

In the above technical solution, the dosage form of the medicine is capsule, liposome, granule, injection, tablet or oral liquid.

The present invention adopts C57BL/6 mice to carry out experiments. In the mouse model induced by isoproterenol, treat with houttuyfonate sodium to observe the improvement effect on cardiac function and cardiac fibrosis, and determine its effect on cardiac fibroblasts. The effect of proliferation ability was determined by houttuyfonate sodium inhibiting fibroblast proliferation, anti-cardiac fibrosis, improving cardiac function, and treating stress cardiomyopathy.

Compared with the prior art, the present invention has the following beneficial effects:

1. Houttuyfonate Sodium has preventive and cardioprotective effects on stress cardiomyopathy; it can reduce cardiac dysfunction caused by stress cardiomyopathy.

2. Houttuyfonate sodium can inhibit myocardial fibrosis, inhibit the proliferation of cardiac fibroblasts, and inhibit the expression of fibrosis marker proteins.

3. Houttuyfonate sodium has a wide range of sources, and has good clinical application prospects in the prevention and treatment of cardiac hypofunction and myocardial fibrosis caused by stress cardiomyopathy.

Description of drawings

Figure 1 shows that Houttuyfonate Sodium improves cardiac function in stress cardiomyopathy.

Fig. 2 is houttuyfonate sodium improving cardiac morphological structure in stress cardiomyopathy.

Fig. 3 is the cytotoxicity test result of houttuyfonate sodium to fibroblasts, ISO: isoproterenol; SH: houttuyfonate sodium.

Fig. 4 is the experimental result of Houttuyfonate Sodium inhibiting the cell proliferation of isoproterenol-induced fibroblasts, ISO: Isoproterenol; SH: Houttuyfonate Sodium.

Fig. 5 is that houttuyfonate sodium inhibits the expression result of cardiac fibroblast fibrosis marker protein induced by isoproterenol and transforming growth factor beta, ISO: isoproterenol; SH: houttuyfonate sodium; TGF- β: transforming growth factor beta.

detailed description

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

1. Materials

1.1 Animals

C57BL/6 mice aged 9-10 weeks were provided by the Experimental Animal Center of Xuzhou Medical University, license number for experimental animals: SCXK (Su) 2020-0011, license for use of experimental animals SYXK (Su) 2020-0048.

1.2 Main instruments

Cardiac ultrasound machine (vevo2100, Visualsonics, USA), fluorescence microscope (Olympus B5167 Japan), thermo microplate reader.

1.3 Drugs and reagents

Sodium houttuynate (China Chengdu Purify Technology Development Co., Ltd., CAS No. 83766-73-8, purity 98%), isoproterenol (Sigma Aldrich), CCK8 (Yacoin Biotechnology Co., Ltd., Wuhan, China). HE dye and Masson dye (Sevier Biotechnology Co., Ltd., Wuhan, China).

2. Method

2.1 Stress cardiomyopathy mouse model and treatment

Isoproterenol was dissolved in 0.9% saline before use. Mice were injected subcutaneously with isoproterenol (15 mg/kg/day) or 0.9% saline daily for 2 weeks to induce heart failure. In the early treatment group and the late treatment group, mice were injected with houttuyfonate sodium (100 mg/kg/day) every day for a specific period of time. The animals were randomly divided into four groups (5-10 in each group): (1) control NS (normal saline) group, mice injected with normal saline for 5 weeks; (2) Houttuyfonate sodium control group: control group mice (3) Stress cardiomyopathy: give isoproterenol for 2 weeks, then inject normal saline for 3 weeks; (4) Treatment group: inject isoproterenol and mice on the same day Houttuyfonate sodium, isoproterenol injection lasted for 2 weeks, and Houttuybein sodium injection lasted for 5 weeks. Five weeks after the administration of isoproterenol, echocardiography was performed and heart weight, lung tissue weight, body weight were measured, and heart tissue was stained with HE and Masson.

2.2 Echocardiography

After 5 weeks of mouse treatment, the cardiac function of the mice was assessed by transthoracic echocardiography. During this procedure, mice were lightly sedated with 1%-2% isoflurane and placed in a supine position on a heated platform with a vevo2100 cardiovascular research ultrasound device (Vision Electronics, Inc., USA). Left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) were assessed at the level of the papillary muscle using the M-mode long-axis view, and left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated using the Simpson method . This process is a double-blind experiment.

2.3 Hematoxylin-eosin (HE) staining and Masson staining

Body weight of mice was determined by echocardiography. Mice were then sacrificed. Mouse hearts and lungs were dissected and weighed separately. Hearts were collected for molecular studies of cardiac remodeling. Each heart was divided laterally into 2 sections: one section (upper) was fixed in 4% formalin for 24 hours, embedded in paraffin, and cut into 5 micron thick sections for histological evaluation. HE and Masson staining were performed. The other part (lower part) was immediately frozen in a -80°C freezer for protein analysis. For consistency and ease of comparison between groups, the same portion from each animal was used for analysis in similar experiments. Each section was imaged under a fluorescence microscope.

2.4 Immunohistochemical staining

Paraffin sections of heart tissue were dewaxed, rinsed three times with PBS, carried out antigen retrieval with sodium citrate solution, and then treated with 0.3% Triton-X 100 for 15 minutes. Blocked with 5% bovine serum albumin for 1 hour, then incubated overnight at 4°C with anti-α-SMA (1:200), col1a (1:200). After washing with PBS for 5 minutes × 3 times, it was incubated with secondary antibody (1:100) for 1 hour at room temperature. Wash three times with PBS, then stain with DAPI for nuclei, wash once with PBS, and mount the slides. Observe under a fluorescence microscope.

2.5 Cell proliferation analysis

2.0× ¹⁰³ cells per well were seeded into 96-well culture plates and cultured for 24 hours, and then treated with isoproterenol and houttuyfonate sodium for 24 hours or 48 hours. Cell proliferation ability was assessed using CCK-8 (Abbkine, China) according to the manufacturer's instructions.

2.6 western blot Western blot analysis

Total protein was extracted from cells with RIPA lysis buffer containing PFSM and phosphatase inhibitors. The extract was spun at 4 °C and 12000 rpm for 15 min, and the supernatant was collected and kept at 80 °C. Protein concentration was quantified using BSA. Use 10% sodium dodecyl sulfate (SDS) polyacrylamide gel (loading buffer, RIPA lysate, SDS-PAGE gel preparation kit, purchased from Jiangsu Biyuntian Institute of Biotechnology) for 30 μg/well Protein electrophoresis. After electrophoresis, the gel was directly transferred to a polyvinylidene fluoride (PVDF) membrane (0.45um, microporous). Membranes were blocked with BSA (3%) for 2 hours. Then incubated overnight at 4°C with primary antibodies: anti-Col1a antibody (1:1000), anti-a-SMA antibody (1:1000), anti-MMP2 antibody (1:1000), anti-TIMP2 antibody (1:1000) and anti-GAPDH (1:5000). After washing three times (5 min/time) with TBST, the cells were incubated with the secondary antibody for 2 hours. Blots were visualized by ECL substrates using Tanon-4800 Multifunctional Imaging System (Tanon, China).

3. Results

3.1 Houttuyfonate Sodium Alleviates Isoproterenol-induced Cardiac Dysfunction in Mice

Intermittent subcutaneous injection of isoproterenol induces cardiac dysfunction in mice. The animals were randomly divided into four groups: control NS group, control houttuyfonate sodium group, isoproterenol-induced cardiomyopathy group, and treatment group. Compared with the NS group, there was no change in the heart function of the mice receiving houttuybein sodium injection for five weeks (control houttuybein sodium group). Compared with the two control groups, isoproterenol-treated mice had impaired cardiac function, characterized by significantly lower LVEF% (left ventricular ejection fraction) and LVFS% (shortened left ventricular ejection), LVEDV (left ventricular End-diastolic volume) and LVESV (left ventricular systolic volume) increased (Fig. 1A-Fig. 1E), heart weight/body weight ratio (HW/BW) and lung weight/body weight ratio (LW/BW) after isoproterenol administration significantly increased (Fig. 1F, Fig. 1G).

In a therapeutic model, houttuyfonate sodium significantly improved the HW/BW ratio, LW/BW ratio, and cardiac function in mice compared with the isoproterenol-cardiomyopathy group.

3.2 Houttuyfonate Sodium Alleviates Cardiac Fibrosis Induced by Isoproterenol in Mice

H&E staining of cardiac tissue showed that isoproterenol injection induced cardiomyocyte hypertrophy. Masson staining of mouse heart sections revealed that isoproterenol treatment resulted in a marked increase in cardiac fibrosis. In the therapeutic model, Houttuyfonate sodium treatment significantly reduced cardiomyocyte hypertrophy and myocardial fibrosis (Fig. 2A, Fig. 2B). Immunofluorescence analysis revealed that isoproterenol administration increased the expression levels of fibrosis markers, including Col1a, while houttuyfonate sodium administration significantly decreased the expression of cardiac fibrosis markers in the treated model. In a therapeutic model, administration of houttuyfonate slightly attenuated cardiac fibrosis. These results indicated that houttuyfonate sodium inhibited isoproterenol-induced fibrosis, could effectively prevent cardiac remodeling and improve cardiac function (Fig. 2C).

3.3 Houttuyfonate sodium inhibits the proliferation of cardiac fibroblasts induced by isoproterenol

Cardiac fibroblasts play a crucial role in cardiac fibrosis in heart failure. To better understand the mechanism of houttuyfonate sodium treatment, isoproterenol triggers the proliferation of cardiac fibroblasts, mimicking myocardial fibrosis in an animal model of stress cardiomyopathy. As shown in Figure 3A, based on CCK-8 analysis, when cells were treated with 0-50 μM isoproterenol for 24 or 48 hours, it increased the cell survival rate of cardiac fibroblasts in a dose-dependent manner (*p<0.05 , Figure 3A). The increased cell viability began to decline upon treatment with 100 micromolar isoproterenol. After 48 hours of treatment with 200 micromolar isoproterenol, cell viability decreased to normal (p>0.05, Figure 3A). This result indicated that the effect of excessive concentration of isoproterenol on fibroblasts for a long time would lead to decrease of cell viability and cell death. Meanwhile, in order to evaluate the cytotoxicity of Houttuyfonate Sodium, cardiac fibroblasts were treated with different Houttuyfonate Sodium concentrations (0-200 micromolar) within 24 and 48 hours, and there was no significant change in cell viability (p>0.05 , Figure 3B).

Next, 10 micromolar isoproterenol-induced cardiac fibroblasts were treated with houttuyfonate sodium (0, 10, 20, 50, and 100 micromolar) for 48 hours. Houttuyfonate sodium treatment at concentrations of 50 μM and 100 μM inhibited cardiac fibroblast proliferation in a dose-dependent manner (p<0.05, FIG. 4A ).

Finally, in order to evaluate whether 100 micromolar houttuyfonate sodium has a significant inhibitory effect on the proliferation of cardiac fibroblasts induced by different concentrations of isoproterenol. After treating cardiac fibroblasts induced by different concentrations of isoproterenol (10-100 micromolar) with 100 micromolar houttuyfonate sodium for 48 hours, there was no significant difference in cell viability between all treatment groups and the normal group (p >0.05, FIG. 4B), indicating that 100 micromole houttuyfonate sodium has a significant inhibitory effect on the proliferation of different degrees of cardiac fibroblasts.

3.4 Houttuyfonate Sodium Inhibits the Expression of Cardiac Fibroblast Fibrosis Marker Proteins Induced by Isoproterenol and Transforming Growth Factor β

In order to further confirm that houttuyfonate sodium inhibits isoproterenol-induced fibrosis of cardiac fibroblasts. Related fibrosis marker proteins were also detected by protein analysis: collagen type I (Col1a), alpha smooth muscle actin (a-SMA), matrix metalloproteinase 2 (MMP2) and tissue inhibitor of metalloproteinase 2 (TIMP2), These proteins represent the activation of fibroblasts into myofibroblasts, and also represent the level of extracellular matrix, which can reflect the degree of fibrosis. 100 μM houttuyfonate sodium decreased the expression of Col1a, a-SMA, MMP2 and TIMP2 in isoproterenol-induced cardiac fibroblasts (*p<0.05, **p<1.01, ***p<2.001, ***p<0.0001, Figure 5A-E). It is well known that transforming growth factor β (TGF-β) plays an important role in myocardial fibrosis caused by various heart diseases. Therefore, the inventors also tested whether houttuyfonate sodium can inhibit the fibrosis of cardiac fibroblasts induced by transforming growth factor β. The study found that 100 micromolar houttuyfonate sodium significantly inhibited the expression of Col1a, a-SMA, MMP2 and TIMP2 in cardiac fibroblasts induced by transforming growth factor β (*p<0.05, **p<1.01, ***p <2.001, ***p<0.0001, Figure 5A-E). This shows that houttuyfonate sodium can significantly inhibit the activation and secretion of fibroblasts under pathological conditions, inhibit the remodeling of extracellular matrix, and thereby inhibit fibrosis.

In summary, the present invention has been proved by experiments: (1) houttuyfonate sodium has preventive and cardioprotective effect on stress cardiomyopathy; (2) can reduce cardiac dysfunction caused by stress cardiomyopathy. (3) It can inhibit myocardial fibrosis, inhibit fibroblast proliferation, and inhibit the expression of fibrosis marker proteins. It has a good clinical application prospect in the prevention and treatment of cardiac hypofunction and myocardial fibrosis caused by stress cardiomyopathy.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone familiar with the technical field within the technical scope disclosed in the present invention, whoever is within the spirit and principles of the present invention Any modifications, equivalent replacements and improvements made within shall fall within the protection scope of the present invention.

Claims (6)

1. Application of Houttuyfonate Sodium in the preparation of drugs for the prevention and treatment of stress cardiomyopathy. 2. Application of Houttuyfonate Sodium in the preparation of drugs for the prevention and treatment of myocardial fibrosis. 3. application according to claim 2, is characterized in that, described medicine suppresses the cardiac fibroblast proliferation that isoproterenol induces, suppresses the cardiac fibroblast Col1a, α-SMA, MMP2 and Expression of TIMP2. 4. The application according to claim 1 or 2, characterized in that the medicament comprises an effective dose of Houttuyfonate Sodium and a pharmaceutically acceptable carrier. 5. The application according to claim 1 or 2, characterized in that the dosage of said houttuyfonate sodium is 100 mg/kg·day. 6. The application according to claim 4, wherein the dosage form of the medicine is capsule, liposome, granule, injection, tablet or oral liquid.

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