Application of motherwort granules in preparation of medicine for improving cardiac hypertrophy and / or heart failure

Abstract

The invention relates to the technical field of cardiology medical treatment, in particular to application of motherwort granules in preparation of a medicine for improving cardiac hypertrophy and / or heart failure. The invention aims to provide application of motherwort granules in preparation of drugs for improving cardiac hypertrophy and heart failure induced by pressure load. Experimental analysis shows that the motherwort granules can effectively inhibit cardiac hypertrophy and heart failure induced by pressure load, and a new medicine and a new treatment scheme are provided for clinical treatment of patients with the diseases. The application proves the effect of the motherwort granules in myocardial hypertrophy and heart failure for the first time, and meanwhile, the motherwort granules as a traditional Chinese medicine for promoting blood circulation to remove blood stasis have smaller side effects than other existing cardiovascular medicines, and are suitable for being popularized and applied in preparation of treatment medicines. The application can provide a new treatment means for treating cardiac hypertrophy and improving heart failure.

Description

Technical Field

[0001] This application relates to the technical field of cardiology medicine, and particularly to the use of Yimu Granules in the preparation of a medicament for improving myocardial hypertrophy and / or heart failure. Background Art

[0002] According to the latest data of the "Report on Cardiovascular Health and Diseases in China", the prevalence and mortality of cardiovascular diseases in China continue to rise. Cardiovascular diseases cause more than 45% of the deaths from diseases among residents, ranking first, higher than tumors and other diseases, seriously threatening human health, and bringing huge social and economic burdens, posing a severe public health challenge.

[0003] Myocardial hypertrophy is an adaptive response of cardiomyocytes to persistent increased load. Initial myocardial hypertrophy has certain compensatory significance. However, with the development of the disease, this process of myocardial thickening and subsequent myocardial remodeling may ultimately lead to the occurrence of heart failure, becoming an independent and key risk factor that significantly promotes the incidence and mortality of cardiovascular diseases. Recent research progress has further emphasized the important role of myocardial hypertrophy in the process of cardiovascular diseases and pointed out that early identification and intervention are of great significance for improving the prognosis of patients and reducing the disease burden. Currently, there is no unified drug for the treatment of myocardial hypertrophy complicated with heart failure, and usually, it is necessary to prescribe the right medicine according to different causes.

[0004] Yimu Granules (abbreviated as: YMKL) are developed by Zhuzhou Qianjin Pharmaceutical Co., Ltd., which can activate blood circulation and regulate menstruation, promote qi flow and relieve pain, and are mainly used for the treatment of diseases such as qi stagnation and blood stasis, irregular menstruation, dysmenorrhea, and abdominal pain due to postpartum blood stasis. Its main components are motherwort, angelica, chuanxiong, and costus root, and it has been on the market.

[0005] Currently, there is no relevant report on the use of Yimu Granules in the treatment of myocardial hypertrophy and heart failure. Summary of the Invention

[0006] This application provides the use of Yimu Granules in the preparation of a medicament for improving myocardial hypertrophy and / or heart failure.

[0007] The purpose of this application is to provide the use of Yimu Granules in the preparation of a medicament for improving myocardial hypertrophy and heart failure induced by pressure load. Through experimental analysis, it can be known that Yimu Granules can effectively inhibit myocardial hypertrophy and heart failure induced by pressure load, providing a new drug and a new treatment plan for the clinical treatment of patients with such diseases.

[0008] This application provides the use of Yimu Granules in the preparation of a medicament for improving myocardial hypertrophy and / or heart failure.

[0009] Optionally, the myocardial hypertrophy is induced by the pressure load of aortic arch constriction surgery.

[0010] Optionally, the motherwort granules inhibit the hypertrophy of cardiomyocytes and the increase of heart weight index by inhibiting the expression of Anp, Bnp, and Myh7.

[0011] Optionally, the heart failure is induced by the pressure load of aortic arch constriction surgery.

[0012] Optionally, the motherwort granules can increase the left ventricular ejection fraction and the fractional shortening of the short axis.

[0013] Optionally, the dosage of the motherwort granules in the drug for improving myocardial hypertrophy and / or heart failure induced by the pressure load of aortic arch constriction surgery is 2 - 8 g / kg / day.

[0014] In summary, the present application includes at least one of the following beneficial technical effects:

[0015] By intragastric administration of motherwort granules to mice with pressure load-induced myocardial hypertrophy, it is found that the motherwort granules can significantly inhibit myocardial hypertrophy in mice and delay the occurrence of heart failure, and can provide new drugs and new treatment regimens for the clinical treatment of patients with myocardial hypertrophy and heart failure.

[0016] The present application demonstrates for the first time the role of motherwort granules in myocardial hypertrophy and heart failure. At the same time, as a traditional Chinese medicine for promoting blood circulation and removing blood stasis, the motherwort granules have relatively small side effects compared with other existing cardiovascular drugs and are suitable for the preparation of therapeutic drugs for popularization and application. The present application can provide a new treatment means for treating myocardial hypertrophy and improving heart failure. Description of the Drawings

[0017] Figure 1 The detection results of echocardiography of mice in each group in the preventive experiment ((A) Representative echocardiogram of mice in each group after 8 weeks; (B) Measurement results of left ventricular ejection fraction (LVEF); (C) Measurement results of left ventricular fractional shortening (LVFS)).

[0018] Figure 2 The morphological observation and staining results of the hearts of mice in each group in the preventive experiment ((A) From top to bottom are the gross photos of the mice's hearts during material collection, the representative HE staining diagram and WGA staining diagram of the cross-section of the cardiac papillary muscle; (B) The ratio of heart weight to tibia length; (C) The quantitative results of the cross-sectional area of cardiomyocytes in the WGA staining diagram).

[0019] Figure 3 The statistical results of fluorescence quantitative PCR of three cardiac hypertrophy marker genes, Anp, Bnp, and Myh7, in the preventive experiment.

[0020] Figure 4Detection results of echocardiography and survival rate analysis results of mice in each group in the therapeutic experiment ((A) Representative echocardiogram of mice at 6 weeks and 10 weeks; (B) Survival curve of mice after intervention starting from 6 weeks; (C) Measurement results of left ventricular ejection fraction (LVEF); (D) Measurement results of left ventricular fractional shortening (LVFS)). Detailed implementation manners

[0021] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this term belongs.

[0022] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0023] In the present application, the endpoints and any values in the disclosed ranges are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0024] In the present application, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present application, but does not exclude other aspects of the content.

[0025] To make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application and should not be construed as a limitation to the present application.

[0026] For those not specified with specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specified with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] The present application will be further described in detail below in combination with the embodiments and the detection results.

[0028] The following examples verified that Yimu Granules can significantly inhibit myocardial hypertrophy in mice and delay the onset of heart failure.

[0029] (I) Experimental reagents

[0030] 1. The Yimu Granules used were in the form of granules, produced by Zhuzhou Qianjin Pharmaceutical Co., Ltd. When used, the Yimu Granules were formulated into an aqueous solution of 0.8 g / mL.

[0031] 2. Ready-to-use tribromoethanol was purchased from Nanjing Aibei Biotechnology Co., Ltd., and the final concentration of tribromoethanol was 20 mg / mL.

[0032] (II) Experimental animals

[0033] Male C57BL / 6 mice (8 - 10 weeks old) were purchased from the Animal Department of Peking University Health Science Center.

[0034] (III) Experimental methods

[0035] 1. Establishment of the TAC model

[0036] The transverse aortic constriction method was used to construct a myocardial hypertrophy model in mice. This method does not require the assistance of a ventilator and is a minimally invasive surgery that can effectively reduce the harm to mice. The specific steps are as follows:

[0037] (1) Preparation before surgery: All surgical instruments required were sterilized by high-temperature and high-pressure one day before the surgery.

[0038] (2) Before the start of the surgery, the neck skin of the mice was depilated with depilatory cream in advance. The mice were anesthetized by a single intraperitoneal injection of 450 μL of tribromoethanol, placed supine on a clean operating table, and their limbs were fixed with tape.

[0039] (3) The depilated area was disinfected with iodophor cotton balls. The skin of the mice was longitudinally incised at the midline of the neck and chest with a straight ophthalmic scissors, and the connective tissue and the muscles on both sides of the tracheal midline were bluntly separated with a curved ophthalmic forceps. One side of the sternocleidomastoid muscle was clamped with forceps, and the sternum was cut open longitudinally from the middle with a microsurgical scissors (the cutting position did not exceed the second rib). After opening the chest, white thymus tissue and mediastinal fat could be seen. The thymus was gently torn apart from the middle with microsurgical forceps and then fixed on both sides with a rib retractor to expose the aortic arch.

[0040] (4) Threading: A threading hook with a 6-0 non-absorbable suture was slowly inserted downward from under the transverse aortic arch between the brachiocephalic trunk and the left common carotid artery at the lower edge of the trachea. When it entered about 2 mm, the threading hook was rotated 90° towards the head end of the mouse with the transverse aortic arch as the center and slowly pulled out. After holding the thread end with forceps, the thread end was slowly pulled out from under the transverse aortic arch to complete the threading.

[0041] (5) Ligation: Place a blunt 27-gauge needle bent at 90° beside the aortic arch, and tie the suture with two micro forceps (usually make 3 single knots). After ligation, slowly remove the 27-gauge needle with the assistance of micro forceps. After successful aortic arch stenosis, a significantly enhanced pulsation of the right common carotid artery can be observed.

[0042] (6) Suture: Suture the sternum with an absorbable suture with a needle (only one suture is needed here), then suture the skin (about 3 - 4 sutures), and disinfect the wound with an iodophor cotton ball. After the operation, place the mouse on a heating pad and wait for it to wake up.

[0043] In the sham operation group, except for not ligating the aortic arch, the rest of the operation process is the same as that in the TAC group.

[0044] Example

[0045] Example 1

[0046] This example explored the effects of Yimu Granules on myocardial hypertrophy and heart failure in mice in a preventive experiment.

[0047] The specific process is as follows:

[0048] I. Grouping and administration

[0049] 1. Grouping: The experiment was divided into six groups, namely:

[0050] Sham + double distilled water group (Sham group);

[0051] Sham + Yimu Granules (8 g / kg) group (Sham + YMKL group);

[0052] Surgery + double distilled water group (TAC group);

[0053] Surgery + low-dose Yimu Granules (0.5 g / kg) group (TAC + YMKL LD group);

[0054] Surgery + medium-dose Yimu Granules (2 g / kg) group (TAC + YMKL MD group);

[0055] Surgery + high-dose Yimu Granules (8 g / kg) group (TAC + YMKL HD group).

[0056] 2. Administration method

[0057] Sham group: On the first day after surgery, intragastrically administer double distilled water (body weight * 10 μL) / day for 8 consecutive weeks;

[0058] Sham + YMKL group: On the first day after surgery, intragastrically administer 0.8 g / mL YMKL aqueous solution (body weight * 10 μL) / day for 8 consecutive weeks;

[0059] TAC group: On the first day after surgery, gavaged with double-distilled water (body weight * 10 μL) / day for 8 consecutive weeks;

[0060] TAC + YMKL LD group: On the first day after surgery, gavaged with 0.05 g / mL YMKL aqueous solution (body weight * 10 μL) / day for 8 consecutive weeks;

[0061] TAC + YMKL MD group: On the first day after surgery, gavaged with 0.2 g / mL YMKL aqueous solution (body weight * 10 μL) / day for 8 consecutive weeks;

[0062] TAC + YMKL HD group: On the first day after surgery, gavaged with 0.8 g / mL YMKL aqueous solution (body weight * 10 μL) / day for 8 consecutive weeks.

[0063] Example 2

[0064] This example explored the effects of Yimu Granules on myocardial hypertrophy and heart failure in mice in a therapeutic experiment.

[0065] The specific process is as follows:

[0066] I. Grouping and administration

[0067] 1. Grouping: The experiment was divided into three groups, namely:

[0068] Sham operation + double-distilled water group (Sham group);

[0069] Surgery + double-distilled water group (TAC group);

[0070] Surgery + Yimu Granules (8 g / kg) group (TAC + YMKL group).

[0071] 2. Administration method

[0072] Sham group: Starting from the 7th week after surgery, gavaged with double-distilled water (body weight * 10 μL) / day for 4 consecutive weeks;

[0073] TAC group: Starting from the 7th week after surgery, gavaged with double-distilled water (body weight * 10 μL) / day for 4 consecutive weeks;

[0074] TAC + YMKL group: Starting from the 7th week after surgery, gavaged with 0.8 g / mL YMKL aqueous solution (body weight * 10 μL) / day for 4 consecutive weeks.

[0075] Detection test

[0076] The following detections were carried out on Example 1 (preventive experiment) and Example 2 (therapeutic experiment) respectively.

[0077] I. Detection method

[0078] 1. Echocardiogram detection

[0079] (1) Preventive experiment - Example 1

[0080] Eight weeks after drug administration, a Visual Sonics Vevo 2100 small animal ultrasonic imaging system was used to evaluate cardiac function by echocardiogram.

[0081] The mice were depilated on the chest, anesthetized with isoflurane, and fixed in the supine position. A probe was used to collect left ventricular long-axis sectional images and left ventricular short-axis M-mode ultrasonic images to evaluate the cardiac function of the mice. Echocardiogram continuously recorded images of 3 complete cardiac cycles and measured the average values of 3 cardiac cycles as the detection values.

[0082] Cardiac systolic function detection indexes include left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS). The heart rate of the mice was controlled at 500 ± 30 beats per minute.

[0083] (2) Therapeutic experiment - Example 2

[0084] Six weeks after TAC surgery, the mice were subjected to cardiac ultrasound detection, and then divided into three groups for intervention according to LVEF and LVFS. Four weeks after the intervention, the mice were subjected to cardiac ultrasound detection again to evaluate the changes in cardiac systolic function.

[0085] 2. Determination of the ratio of heart weight to body weight and the ratio of heart weight to tibia length

[0086] At the end of the experiment, the body weight (BW) of the mice was measured and blood was collected by enucleating the eyeballs. After the mice were decapitated and the chest cavity was opened, perfusion was performed with PBS. After the perfusion was completed, the heart tissue was removed, dried, and weighed (HW). Then, half of the myocardial tissue was fixed (4% paraformaldehyde) for pathological paraffin embedding and sectioning; the other half of the myocardial tissue samples were stored in a -80°C refrigerator for detecting protein and RNA expression. The tibia of the mice was taken and the length was measured with a vernier caliper.

[0087] 3. Morphological observation of myocardial tissue and determination of myocardial fibrosis area

[0088] After the heart was fixed in 4% paraformaldehyde solution for 24 h, it was paraffin-embedded and sectioned (thickness 5 μm). Then, hematoxylin and eosin (HE) staining and wheat germ agglutinin (WGA) staining were performed respectively. The morphology of myocardial tissue was observed under an optical microscope. After photographing the WGA staining map under a fluorescence microscope, the size of cardiomyocytes was measured using Image J software.

[0089] 4. Real-time fluorescence quantitative PCR

[0090] About 5-10 mg of myocardial tissue was taken, and the RNA of heart tissue was extracted using an RNA extraction kit (Aikerui Biotech, product number: AG21017). The concentration and purity of the RNA sample were detected using NanoDrop 2000. Reverse transcription was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Quanshijin Biotech, product number: AT311-03). Real-time fluorescence quantitative PCR was performed using Top Green qPCR SuperMix Top Green fluorescence quantitative PCR kit (Quanshijin Biotech, AQ131-01) on an Agilent AriaMx machine.

[0091] The primer sequences for real-time fluorescence quantitative PCR are shown in Table 1.

[0092] Table 1 Primer sequences for real-time fluorescence quantitative PCR

[0093]

[0094]

[0095] 5. Statistical methods

[0096] Statistical analysis was performed using GraphPad Prism8 software, and the data were statistically analyzed using T-Test. The data were expressed as mean ± SEM, and when P < 0.05, it indicated that the difference was statistically significant.

[0097] II. Experimental results

[0098] 1. Preventive experiment - Example 1

[0099] (1) YMKL improved myocardial hypertrophy and decreased cardiac systolic function caused by pressure load in mice

[0100] After 8 weeks of drug administration, echocardiography was performed on each group of mice, and the results are as Figure 1 shown.

[0101] By Figure 1It can be seen that compared with the Sham group, obvious myocardial hypertrophy and ventricular dilation occurred in the hearts of mice in the TAC group (see Figure 1 A), accompanied by a significant decrease in cardiac systolic function (significant decreases in LVEF% and LVFS%, see Figure 1 B and Figure 1 C). After intervention with YMKL MD dose and YMKL HD dose, myocardial hypertrophy and ventricular dilation in TAC mice were significantly inhibited, and cardiac systolic function was significantly improved (LVEF% and LVFS% were significantly increased compared with the TAC group).

[0102] After taking samples from the mouse hearts, the whole hearts were photographed, and HE staining and WGA staining of the heart cross-sections were performed. The results are as Figure 2 shown.

[0103] It can be Figure 2 seen that compared with the Sham group, the hearts of mice in the TAC group were significantly hypertrophied (see Figure 2 A), the cross-sectional areas of the cardiac papillary muscles and myocardial cells were significantly increased (see Figure 2 A and Figure 2 C), and the ratio of heart weight to mouse tibia length was significantly increased (see Figure 2 B). Compared with the TAC group, after intervention with YMKL MD and HD doses, cardiac hypertrophy in mice was significantly alleviated, the cross-sectional area of the cardiac papillary muscles was significantly decreased, and the cross-sectional area of myocardial cells was significantly decreased under the intervention of all three YMKL doses.

[0104] (2) YMKL down-regulates the expression of genes related to pressure overload-induced cardiac hypertrophy

[0105] After extracting RNA from the mouse heart tissues, the expression changes of the cardiac hypertrophy marker genes Anp, Bnp, and Myh7 in each group of mice were detected by qPCR. The results are as Figure 3 shown.

[0106] It can be Figure 3 seen that compared with the sham group, the expressions of Anp, Bnp, and Myh7 in the hearts of mice in the TAC group were all significantly increased, and after intervention with YMKL MD and HD doses, the expressions of Anp, Bnp, and Myh7 were all significantly down-regulated.

[0107] The above experimental results showed that 8 weeks after TAC surgery, obvious hypertrophy and dilation occurred in the hearts of mice, the left ventricular ejection fraction and fractional shortening decreased, the cardiac systolic function was impaired, and the cardiomyocytes became significantly larger. After intervention with YMKL, the left ventricular ejection fraction and fractional shortening of the mice were significantly higher than those in the TAC group, and the cardiomyocytes became significantly smaller. Heart tissues were taken for RNA extraction, and the mRNA expression changes of the hypertrophy marker genes Anp, Bnp, and Myh7 were detected by quantitative fluorescence PCR. The results showed that the expressions of Anp, Bnp, and Myh7 in the TAC group increased significantly, and the expressions of Anp, Bnp, and Myh7 were significantly lower in the YMKL intervention group than in the TAC group. This indicated that YMKL had a certain degree of protective effect on the cardiac pressure overload model caused by TAC surgery.

[0108] 2. Therapeutic experiment - Example 2

[0109] Six weeks after TAC surgery, echocardiography was performed on each group of mice, and the results are as Figure 4 shown.

[0110] As Figure 4 can be seen, compared with the sham group, obvious myocardial hypertrophy and ventricular dilation had occurred in the hearts of TAC mice at 6 weeks, and the cardiac systolic function had also decreased significantly, specifically manifested as significant decreases in LVEF and LVFS (see Figure 4 A, Figure 4 C, and Figure 4 D).

[0111] According to the LVEF and LVFS values at TAC 6 weeks, the mice in the TAC group were grouped for drug administration. Four weeks after drug administration (10 weeks after TAC surgery), echocardiography and survival rate analysis were performed on each group of mice. The results showed that after four weeks of drug administration, no deaths were found in the 8 mice in the Sham group; among the 11 mice in the TAC group, 3 mice died at the end of the experiment; among the 11 mice in the TAC + YMKL group, 1 mouse died (see Figure 4 B). The echocardiogram results showed that the ventricles of the mice in the TAC group further dilated and the cardiac systolic function further decreased at TAC 10 weeks (see Figure 4 A, Figure 4 C, and Figure 4 D). After 4 weeks of treatment with YMKL, it could prevent further hypertrophy of the mouse heart and further decline of cardiac systolic function, showing obvious improvement compared with the TAC group.

[0112] The above experimental results showed that obvious ventricular dilation and myocardial hypertrophy occurred in the hearts of mice 6 weeks after TAC surgery. The left ventricular ejection fraction and fractional shortening both decreased, and the cardiac systolic function was impaired. The ventricles further dilated at the 10th week, and the left ventricular ejection fraction and fractional shortening further decreased. Continuous administration of YMKL for 4 weeks starting from 6 weeks later significantly improved the above changes. It was proved that after myocardial hypertrophy, ventricular dilation and decreased cardiac contractility caused by pressure overload in mice, continuous administration of YMKL could inhibit further myocardial hypertrophy and further decline of cardiac function.

[0113] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit them; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of each embodiment of this application.

Claims

1. Use of motherwort granules in the preparation of a medicament for improving myocardial hypertrophy and / or heart failure.

2. The application according to claim 1, characterized in that, The myocardial hypertrophy is induced by the pressure load of aortic arch constriction surgery.

3. The application according to claim 1, wherein The motherwort granules inhibit the hypertrophy of cardiomyocytes and the increase of heart weight index by inhibiting the expression of Anp, Bnp, and Myh7.

4. The application according to claim 1, characterized in that, The heart failure is induced by the pressure load of aortic arch constriction surgery.

5. The application according to claim 1, characterized in that, The motherwort granules can increase the left ventricular ejection fraction and the fractional shortening of the short axis.

6. The application according to claim 1, wherein The dosage of the motherwort granules in the medicament for improving myocardial hypertrophy and / or heart failure induced by the pressure load of aortic arch constriction surgery is 2 - 8 g / kg / day.

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