CN112156091A - Application of hispidulin in preparation of medicine for treating and/or preventing cardiovascular diseases - Google Patents

Abstract

The invention discloses an application of hispidulin in preparing a medicament for treating and/or preventing cardiovascular diseases. The hispidulin provided by the invention solves the problems that effective prevention and treatment aiming at heart failure are not carried out in the prior art and adverse reactions exist in clinical drug therapy, and provides a new idea and a specific application for preventing and treating heart failure which is a serious cardiovascular disease. The invention provides a new action mechanism for preventing and treating myocardial hypertrophy and heart failure by using the hispidulin from the aspect of molecular, and further provides a new basis for the development and utilization of bioactive components of Chinese traditional Chinese herbal medicine, namely the artemisia argyi and the like, and the invention also has important significance for the effective and reasonable utilization of the Chinese herbal medicine in the field of heart failure. And provides a new strategy for the development of clinical target spots for drug treatment of myocardial hypertrophy and heart failure.

Description

Application of hispidulin in preparation of medicine for treating and/or preventing cardiovascular diseases

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of hispidulin in preparing a medicine for treating and/or preventing cardiovascular diseases.

Background

Heart failure is a common manifestation of the development of cardiovascular diseases (such as hypertension, coronary heart disease, various cardiomyopathies, valvular heart diseases, etc.) to the terminal stage. Ventricular remodeling, including myocardial hypertrophy and fibrosis, is a central link in the progression of heart failure. In the early stage of cardiovascular diseases, pathological factors such as myocardial ischemia, cardiac preload/afterload increase, myocardial lesion and the like act on the heart, and firstly, compensatory hypertrophy of the myocardium is caused to ensure the blood pumping function of the heart and maintain the stability of the heart function, which is mainly characterized in that the volume of myocardial cells is increased, the weight of the heart is increased, the wall of a chamber is thickened, a signal transduction path related to the hypertrophy of the myocardium is activated, the synthesis of protein is increased, and the expressions of embryonic genes Atrial Natriuretic Peptide (ANP) and B-type Natriuretic Peptide (BNP) are obviously increased. However, as the condition progresses, persistent myocardial hypertrophy promotes the progression of myocardial fibrosis, causing apoptotic necrosis of myocardial cells, corresponding thinning of the ventricular wall, enlargement of the cardiac chamber, decline of myocardial compliance, decline of cardiac output, and finally heart failure. Therefore, the cardiac hypertrophy and the cardiac fibrosis are used as core links for the occurrence and the development of the heart failure, and the inhibition of the progress of the cardiac hypertrophy and the cardiac fibrosis has important significance for preventing and treating the heart failure.

The heart, as an organ of the body with high energy requirements, requires a sufficient energy supply to ensure its systolic and diastolic function. More than 95% of Adenosine Triphosphate (ATP) in the heart is derived from mitochondrial Oxidative phosphorylation (OXPHOS). ATP is produced via mitochondrial oxidative phosphorylation, which is regulated by a number of aspects including energy substrates, excitation-contraction coupling, and redox balance. When cardiac muscle is hypertrophic, the load around the heart is increased, and the demand for heart energy is increased. Meanwhile, the level of mitochondrial oxidative phosphorylation is reduced, and ATP synthesis is reduced; in addition, the release of active oxygen by the electron transport chain of mitochondria is increased, the antioxidant system is inhibited, and the oxidative stress is enhanced. The development of myocardial hypertrophy to heart failure is eventually exacerbated by increased energy demand and decreased yield, as well as the occurrence of oxidative stress injury. In previous studies, using 31P magnetic resonance spectroscopy (31P MRS), creatine phosphate was found in the heart of heart failure patients: the ratio of adenosine triphosphate (PCr: ATP) is significantly reduced, indicating an insufficient supply of cardiomyocytes in patients with heart failure, which often suggests Left Ventricular (LV) dysfunction and poor prognosis.

Mitochondria are used as the most important energy-producing organelles of myocardial cells, and the stability of the functions of the mitochondria has important significance for inhibiting the further development of myocardial hypertrophy. Mitochondrial respiratory function was significantly inhibited in the aortoconstriction-induced myocardial hypertrophy model in mice. Through gene and proteome analysis, the expression levels of protein and mRNA of a complex I (NADH dehydrogenase complex), a complex II (succinate dehydrogenase complex), a complex III (cytochrome reductase complex) and a complex IV (cytochrome oxidase complex) related to the mitochondrial electron transport chain are remarkably reduced, so that the mitochondrial electron transport chain activity is inhibited, the mitochondrial ATP synthesis is reduced, and the cardiac function is reduced. Several studies have demonstrated that improving mitochondrial dysfunction can inhibit myocardial hypertrophy and its progression to heart failure. Amipropeptide (SS-31) is used as a mitochondrion targeting antioxidant peptide, and is proved in various animal models, and the amiprepeptide stabilizes the electron transfer function of cytochrome C by combining with cardiolipin, improves the mitochondrion oxidative phosphorylation efficiency, and inhibits the generation of mitochondrion ROS, thereby inhibiting the development of myocardial hypertrophy. Coenzyme Q (coenzyme Q) is used as a fat-soluble electron carrier, can improve mitochondrial electron transfer and ATP synthesis, and clinical researches clearly prove that the coenzyme Q serving as an adjuvant drug for chronic heart failure can reduce the morbidity and mortality of the heart failure. However, although stabilization of mitochondrial function is known to have inhibitory effects on the development of cardiac hypertrophy and heart failure, current methods of treatment of cardiac hypertrophy and heart failure are still largely limited to inhibition of neuroendocrine activation, reduction of myocardial oxygen consumption by lowering ventricular load and reduction of heart rate, and the discovery of drugs directed directly at improving mitochondrial function in the myocardium is very limited. Therefore, the research on new drugs and targets with protective effect on mitochondrial function in myocardium and the deep research on the action mechanism thereof have important significance on the prevention and treatment of myocardial hypertrophy and heart failure.

The hispidulin exists in kiwi fruit, snow lotus, snow grass, wormwood and red sage root, and has a plurality of applications in the fields of tumor and neurology because of various biological effects of oxidation resistance, apoptosis resistance, inflammation resistance, mutation resistance, tumor resistance and the like. However, in the cardiovascular field, there is no research on hispidulin at home and abroad.

Disclosure of Invention

The invention aims to provide application of hispidulin in preparing a medicament for treating and/or preventing cardiovascular diseases. The invention takes a pressure overload mouse induced by aortic constriction as an experimental object, and takes cardiac ultrasound, cardiac weight and size, hematoxylin-eosin (H & E) of a cardiac cross section, wheat germ agglutinin WGA staining, cardiac mitochondrial function and mitochondrial oxidative stress as detection indexes. The results show that the hispidulin has a significant effect of preventing and treating cardiac hypertrophy induced by pressure overload and can significantly improve heart failure as a natural flavonoid compound, and intensive mechanism studies show that the hispidulin can play a role of protecting mitochondrial productivity and inhibiting mitochondrial oxidative stress to protect cardiac hypertrophy and heart failure, and thus the hispidulin plays an important role in the prevention and treatment of cardiovascular diseases such as cardiac hypertrophy and heart failure (fig. 1).

The inventor finds that the hispidulin has a preventive and therapeutic effect on myocardial hypertrophy and heart failure as a natural compound. The hispidulin can be used as a new method for preventing and treating cardiovascular diseases such as myocardial hypertrophy and heart failure.

The above object of the present invention is achieved by the following scheme:

the invention claims the application of hispidulin in preparing medicines for treating and/or preventing cardiovascular diseases.

Preferably, the cardiovascular disease is heart failure.

More preferably, the heart failure is a complex clinical syndrome of any one or more of hypertension, coronary heart disease, cardiomyopathy, or valvular heart disease progressing to a terminal ring.

More preferably, the heart failure is manifested as myocardial hypertrophy.

More preferably, the myocardial hypertrophy is pressure overload induced.

Preferably, hispidulin reduces myocardial hypertrophy.

Preferably, hispidulin improves mitochondrial function.

Hispidulin inhibits cardiac hypertrophy by improving mitochondrial function, and exerts its cardiovascular protective effect.

More preferably, hispidulin inhibits a decrease in ATP levels in hypertrophic heart tissue.

More preferably, hispidulin inhibits the rise of ROS levels in hypertrophic heart tissue.

More preferably, hispidulin inhibits structural abnormalities of mitochondria in hypertrophic heart tissue.

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

the hispidulin provided by the invention solves the problems that the heart failure complex clinical syndrome cannot be effectively prevented and treated in the prior art and adverse reactions exist in clinical drug treatment, and provides a new thought and a specific application for preventing and treating heart failure which is a serious cardiovascular disease.

The invention provides a new action mechanism for preventing and treating myocardial hypertrophy and heart failure for the hispidulin, provides a new basis for the development and utilization of bioactive components of the Chinese traditional Chinese medicine, namely the mugwort, and the like from the aspect of molecular, and has important significance for the effective and reasonable utilization of the Chinese traditional medicine in the field of heart failure. And provides a new strategy for the development of clinical target spots for drug treatment of myocardial hypertrophy and heart failure.

Drawings

FIG. 1 is a functional diagram of hispidulin.

FIG. 2 shows that in vivo experiments, hispidulin inhibits heart failure and increases cardiac function in mice induced by aortic constriction, and the hispidulin is injected into abdominal cavity from one day before operation to 4 weeks after operation, and echocardiography is performed after 4 weeks; (A) echocardiographic images of the hearts of each group of mice; (B) measuring mouse left ventricular end-diastolic diameter (LVEDD) via thoracic echocardiography; (C) measuring mouse left ventricular end-systolic diameter (LVESD) via thoracic echocardiography; (D) measuring the left ventricular posterior wall thickness (LVPWD) of the mouse via a thoracic echocardiogram; (E) the mouse ejection fraction (EF%) was measured by thoracic echocardiography; (F) measuring the left ventricular short axis shortening rate (FS%) of the mouse by a thoracic echocardiogram; p <0.05vs corrresponding sham group, # P <0.05vs DMSO + AB group.

FIG. 3 shows that in vivo experiments, hispidulin inhibits heart failure in mice induced by aortic constriction, and is intraperitoneally injected from one day before operation to 4 weeks after operation; (A) taking a mouse heart picture by white light for 4 weeks after operation; (B) h & E staining of mice heart paraffin sections at 4 weeks post-surgery; (C) WGA staining of mice heart paraffin sections 4 weeks after surgery; p <0.05vs corrresponding sham group, # P <0.05vs DMSO + AB group.

FIG. 4 is a view of the reversal of cardiac mitochondrial structural disorders induced by aortic coarctation with hispidulin intraperitoneally administered one day before the operation to 4 weeks after the operation; (A) mitochondrial images in heart tissues of four groups of mice under the magnification of 5800X and 26500X by a transmission electron microscope; (B) the area of a single mitochondrion in the heart of four groups of mice; (C) mouse heart mitochondrial aspect ratio 4 weeks post-surgery; p <0.05vs corrresponding sham group, # P <0.05vs DMSO + AB group.

FIG. 5 shows that hispidulin increases mitochondrial ATP synthesis in heart failure, aortoconstriction induces heart failure in mice, hispidulin is administered intraperitoneally 1 day before operation to 4 weeks after operation, a sample of the heart of the mice is collected after 4 weeks, and the kit detects the ATP concentration in cardiomyocytes, corrected by protein concentration, # P <0.05vs corrserving sham group, # P <0.05vs DMSO + AB group.

FIG. 6 shows the effect of hispidulin on Reactive Oxygen Species (ROS) levels in aortic constriction-induced heart failure, and mouse heart specimens were harvested 4 weeks post-surgery, and mouse heart cryosections ROS were labeled with DHE fluorescent probe and each set of images were observed and taken under a fluorescent microscope.

Detailed Description

The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

In order to better understand the essence of the present invention, mice will be used as experimental subjects to illustrate their new use in the pharmaceutical field. A male C57BL/6 mouse with the age of 8-10 weeks is selected, a pressure overload induced myocardial hypertrophy model is established by using Aortic constriction (AB), and a Sham operation group (Sham) is set. When we found that the mice in the aortic coarctation group had significantly increased heart (significantly increased heart weight/body weight ratio and heart weight/tibia length ratio), enlarged heart (increased LVPWD, LVEDD, and LVESD on echocardiogram), and deteriorated cardiac function (decreased EF%, decreased FS%) compared with the sham group, the successful molding was confirmed by taking the heart specimens of the mice after 4 weeks of operation.

Grouping experiments: mice were randomly divided into 4 groups (8-10 per group): (1) DMSO + Sham group, (2) histatin + Sham group (histatin + Sham group), (3) DMSO + AB group, (4) histatin + AB group. The method comprises the steps of dissolving the hispidulin in a DMSO solution, then giving the mice with the hispidulin solution by 20 mg/kg/day through intraperitoneal injection every day, giving the mice with the solvent DMSO solution with the same amount every day as a control group for 4 weeks, and detecting the cardiac structure and the cardiac function by adopting a Vevo2100 high-resolution small animal ultrasonic imaging system.

Example 1 Effect of hispidulin intervention on pressure overload-induced myocardial hypertrophy and Heart failure

First, observation of the basic State of the mouse

1. Experimental methods

Basic status observations were made for each group of mice, including: developmental status, movement, coat, diet, body weight, etc.

2. Results of the experiment

The Sham group mice grew well, had smooth coat and flexible activity, and were fed normally when ingesting water. The AB group mice had reduced, but no significant difference in activity. The condition of the mouse with high plantaginin is improved compared with the aortic coarctation group.

Second, the heart function of the mouse

1. Experimental methods

4 weeks after administration, inhalation anesthesia (1.5-2% isoflurane with 100% O) was given₂Mixed), and a small animal ultrasonic imaging system is adopted for the transthoracic echocardiography detection. The Left ventricular end-diastolic diameter (LVEDD), the ventricular end-systolic diameter (LVESD), the Left ventricular wall thickness (LVPWD), the Left ventricular short axis Shortening rate (FS%) and the Ejection fraction (EF%) of the mouse heart were measured by taking the Left ventricular long axis line M-ultrasonic examination.

2. Results of the experiment

Echocardiography results as shown in figure 2, mice in DMSO + AB group had significantly increased LVEDD, LVESD, and LVPWD, and significantly decreased EF% and FS% compared to DMSO + Sham group; compared with DMSO + AB group, the mice LVEDD, LVESD and LVPWD of the group of the hispidulin + AB group are all obviously reduced, and meanwhile, EF% and FS% are obviously increased, which shows that the hispidulin can obviously inhibit cardiac hypertrophy and cardiac remodeling, improve the cardiac function of the mice and inhibit heart failure.

Size and weight of heart of mouse

1. Experimental methods

After the cardiac function ultrasonic evaluation is completed, the heart and the lung of the mouse are taken and then weighed, and the heart weight to mouse weight ratio (heart weight/body weight, HW/BW) and heart weight to tibia length ratio (heart weight/biological length, HW/TL), lung weight to mouse weight ratio (lung weight/body weight, LW/BW) and lung weight to tibia length ratio (lung weight/biological length, LW/TL) are calculated.

2. Results of the experiment

After 4 weeks of operation, the hearts of the mice were harvested after the completion of the ultrasonic testing, weighed and gross images of the hearts of each group of mice were taken under white light. As shown in fig. 3A and table 1, the AB mice had significantly increased hearts and significantly increased pulmonary congestion compared to the DMSO + Sham group, and significant increases in HW/BW (heart weight/body weight ratio), HW/TL (heart weight/tibia length ratio), LW/BW (lung weight/body weight ratio), and LW/TL (lung weight/tibia length ratio) were examined. The values of HW/BW, HW/TL, LW/BW and LW/TL in the hispidulin + AB group are significantly reduced compared with those in the DMSO + AB group, and the heart and lung are significantly reduced by naked eyes. The suggestion shows that the intervention of the hispidulin can obviously relieve the cardiac hypertrophy of the mice induced by the pressure overload, simultaneously relieve the pulmonary edema and improve the heart failure.

Table 1 inhibitory effect of hispidulin on myocardial hypertrophy and heart failure:

table 1 shows that in vivo experiments, hispidulin inhibits aortic constriction-induced heart failure. The pratensein was injected intraperitoneally one day before surgery to 4 weeks after surgery. Four groups were tested for heart weight/body weight ratio (HW/BW), heart weight/tibia length ratio (HW/TL), lung weight/body weight ratio (HW/BW), lung weight/tibia length ratio (HW/TL). P <0.05vs corrresponding sham group, # P <0.05vs DMSO + AB group.

Pathological morphology of myocardial tissue of mice

1. Experimental methods

After weighing the hearts, a portion of the hearts were transected perpendicular to the long axis of the heart at the middle and embedded in paraffin. Paraffin sections were deparaffinized to water and H & E and WGA staining was performed as per kit instructions to examine the tissue structure of the heart and the extent of cardiomyocyte hypertrophy.

2. Results of the experiment

The results are shown in FIGS. 3A, B and 3C, which show that the ventricular chambers of mice in the DMSO + AB group are significantly enlarged and the cardiomyocytes are significantly enlarged compared to those in the DMSO + Sham group. In the prognosis of the above indexes, compared with the group of hispidulin and AB, the cross-sectional areas of the ventricular cavity and the myocardial cells are obviously reduced, and the myocardial hypertrophy is obviously improved.

Example 2 Effect of hispidulin on mitochondrial function

First, mouse heart mitochondrial morphology

1. Experimental methods

After 4 weeks of surgery, a small piece of fresh mouse heart tissue was trimmed to a 1mm by 3mm cube and fixed by immersion in a 0.1M solution of sodium formate containing 4% paraformaldehyde and 2.5% glutaraldehyde. The samples were cut into 70nm sections using a microtome and stained with lead citrate. Detection of mitochondria in mouse heart sections was performed using a Tecnai G2 Spirit Twin transmission electron microscope. The shots were taken at a magnification of 5800 x and 6,2500 x, respectively, with approximately 20 shots per heart slice. The area and aspect ratio of individual mitochondria were measured using Image J software.

2. Results of the experiment

As a result, as shown in FIG. 4A, structurally abnormal mitochondria appeared in the DMSO + AB group, and the mitochondria became small and round and disorganized, showing a decrease in the ratio of the mitochondrial aspect ratio and the mitochondrial area, and lost the original linear arrangement. Compared with the DMSO + AB group, the hispidulin + AB group has obviously lighter mitochondrial morphology variation, which is represented by increased mitochondrial aspect ratio and increased mitochondrial area (FIGS. 4A, B and 4C), and the results are statistically significant, which indicates that the hispidulin maintains the normal mitochondrial morphology of the mice with cardiac hypertrophy.

Second, ATP level changes in heart tissue of mice

1. Experimental methods

After 4 weeks, fresh heart tissue samples were collected and the Relative Light Unit (RLU) values for each well were determined by chemiluminescence (luminometer) using the luciferase method according to the kit instructions to calculate the corresponding ATP concentrations.

2. Results of the experiment

As shown in fig. 5, cardiac tissue ATP levels were significantly reduced in DMSO + AB mice compared to DMSO + Sham, while cardiac tissue ATP levels were significantly upregulated in hispidulin + AB mice compared to DMSO + AB.

Third, the heart oxidative stress level of the mice

1. Experimental methods

After 4 weeks, fresh cardiac tissue specimens were harvested, transected perpendicular to the long axis of the heart in the middle and embedded with OCT. The cells were sliced from the fundus to the apex at a thickness of 5 μm. DHE staining was performed to assess superoxide anion levels in heart tissue according to kit instructions. DHE fluorescence intensity is positively correlated with intracellular ROS levels. Fluorescence images (excitation wavelength 488nm, emission wavelength 525nm) were taken with a fluorescence microscope. And the fluorescence intensity of each set of cardiac cryosections was analyzed using Image J.

2. Results of the experiment

As shown in FIG. 6, DHE fluorescence of the heart of mice in the DMSO + AB group is obviously increased compared with that in the DMSO + Sham group, and after the treatment of the hispidulin, the ROS level of heart tissues in the hispidulin + AB group is obviously inhibited compared with that in the DMSO + AB group, which is shown as that DHE fluorescence intensity is obviously weakened.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. Use of hispidulin in the preparation of a medicament for the treatment and/or prevention of cardiovascular disease.

2. The use according to claim 1, wherein the cardiovascular disease is heart failure.

3. The use of claim 2, wherein the heart failure is one or more of hypertension, coronary heart disease, cardiomyopathy, or valvular heart disease, complex clinical syndromes that progress to the end-link.

4. Use according to claim 2, wherein the heart failure is manifested as myocardial hypertrophy.

5. The use of claim 2, wherein the myocardial hypertrophy is pressure overload induced.

6. The use of claim 1, wherein hispidulin reduces cardiac hypertrophy.

7. The use of claim 1, wherein hispidulin enhances mitochondrial function.

8. The use of claim 7, wherein the hispidulin inhibits a decrease in ATP levels in hypertrophic heart tissue.

9. The use of claim 7, wherein hispidulin inhibits the increase in ROS levels in hypertrophic heart tissue.

10. The use of claim 7, wherein the hispidulin inhibits mitochondrial structural abnormalities in cardiac tissue.

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