Application of Xinsunning preparation in preparation of medicine for preventing and treating heart failure

Abstract

The invention provides application of a Xinsunning preparation in preparation of a medicine for preventing and treating heart failure, and relates to the technical field of traditional Chinese medicine, the Xinsunning preparation can obviously improve the heart function, and the Xinsunning preparation can be applied in preparation of the medicine for preventing and treating heart failure by improving the heart function.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicines, and in particular to the technology of preparing medicines for preventing and treating heart failure, and specifically to the application of a Xinsuning preparation in preparing medicines for preventing and treating heart failure. Background Art

[0002] Heart failure is a condition in which the heart's pumping function is impaired due to a variety of reasons. The patient's cardiac output is significantly reduced and cannot meet the metabolic needs of the body's tissues and organs. Heart failure is not an independent disease, but the terminal stage of heart disease. According to the location of heart failure, it can be divided into left heart failure, right heart failure, and total heart failure. According to the level of left ventricular ejection fraction, heart failure can be divided into heart failure with reduced ejection fraction and heart failure with preserved ejection fraction. Heart failure can also be divided into acute heart failure and chronic heart failure. The main clinical symptoms of heart failure patients are dyspnea, cough and sputum, hemoptysis, reduced exercise, dizziness, palpitations, fatigue, etc. Some patients may have symptoms such as vomiting, abdominal distension, anorexia, edema, and ascites. Relevant survey data show that the total number of heart failure patients in my country is about 8.7 million, among which the incidence of heart failure in people over 70 years old exceeds 10%, and the mortality rate of patients within 5 years is as high as 50%.

[0003] Traditional treatment methods include drug therapy, non-drug therapy and surgical intervention. In terms of drug therapy, diuretics, ACE inhibitors, beta-blockers, etc. are often used to reduce cardiac load, dilate blood vessels and other ways to exert therapeutic effects. Non-drug treatment includes lifestyle interventions such as limiting salt intake, controlling weight, quitting smoking and limiting alcohol, which are crucial for the recovery of heart failure. In some cases with complicated conditions, surgical treatments such as pacemaker implantation and heart valve repair are also effective means. Studies have found that the pathogenesis of cardiovascular and cerebrovascular diseases, including heart failure, is relatively complex. Because the effective ingredients in Western medicine are too single, and Western medicine may also cause adverse reactions while treating the disease, it is impossible to eliminate the various causes of heart failure in all aspects. Therefore, it is difficult to effectively exert the therapeutic effect by relying solely on Western medicine. In comparison, the advantages of traditional Chinese medicine in treating heart failure are multi-target regulation, gentle treatment, overall body conditioning and individualized treatment, providing patients with more comprehensive and personalized treatment options.

[0004] In terms of traditional Chinese medicine, there is no disease name for heart failure in traditional Chinese medicine. The Golden Chamber states that "water stagnates under the heart, and the severe ones are palpitations, and the mild ones are short of breath" and "the heart water is heavy and has little air". Heart failure is recorded as "heart water". In the Prescriptions for Emergencies, it is recorded that "heart failure is hidden", and the name "heart failure" is proposed. According to its symptoms, heart failure can be classified into the categories of "heart water", "heart failure", "suspended drink", "asthma" or "edema" in traditional Chinese medicine. The existing Chinese medicine compositions for the treatment of heart failure are mainly treated with the ideas of promoting blood circulation and removing blood stasis, regulating qi and relieving pain. Although they have certain therapeutic effects, they are slow to take effect due to the preparation or prescription composition, and there are still problems such as unsatisfactory long-term efficacy and inability to cure, and the treatment mechanism has not been explored. In addition, drugs for the treatment of cardiovascular and cerebrovascular diseases such as heart failure have limited clinical applications due to side effects, and poor prognosis and high mortality of patients are still the focus and difficulty of clinical research. Therefore, it is of great significance to find new drugs for the prevention and / or treatment of heart failure. Summary of the invention

[0005] In view of the above, the present invention aims to provide a drug for preventing and treating heart failure, which at least solves one of the technical problems of preventing and treating heart failure to a certain extent. Specifically, the present invention proposes the use of the Xinsuning preparation in the preparation of drugs for preventing and treating heart failure, mainly for the preparation of drugs for preventing and treating doxorubicin-induced heart failure.

[0006] In view of the above technical problems, the present invention adopts the following technical solutions:

[0007] The invention discloses an application of the Xinsuning preparation in preparing medicines for preventing and treating heart failure.

[0008] It is further defined that the Xinsuning preparation is used in the preparation of drugs for preventing and treating heart failure by improving cardiac function.

[0009] It is further defined that the improvement of cardiac function includes increasing cardiac ejection fraction, improving left ventricular minor axis shortening rate, reducing left ventricular end-systolic / diastolic inner diameter, reducing left ventricular end-systolic / diastolic anterior wall thickness and / or reducing left ventricular end-systolic / diastolic posterior wall thickness.

[0010] It is further defined that the Xinsuning preparation is used in the preparation of drugs for preventing and treating heart failure by increasing the number of myocardial cell mitochondria.

[0011] It is further defined that the Xinsuning preparation is used in the preparation of drugs for preventing and treating heart failure by improving myocardial structure.

[0012] It is further defined that the improvement of myocardial structure refers to improving the changes induced by doxorubicin on myocardial tissue morphology.

[0013] It is further defined that the myocardial tissue morphology includes the degree of myocardial cell edema, the degree of myocardial cell inflammatory infiltration and the degree of myocardial cell arrangement.

[0014] It is further defined that the Xinsuning preparation is used in the preparation of a drug for preventing and treating doxorubicin-induced heart failure.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The application of the Xinsuning preparation of the present invention in the preparation of drugs for the prevention and treatment of heart failure, wherein the Xinsuning preparation treatment group of the present invention can significantly improve the cardiac ejection fraction of mice, significantly improve the short axis shortening rate of the left ventricle of mice, significantly reduce the left ventricular end-systolic inner diameter, significantly reduce the left ventricular end-diastolic inner diameter, significantly reduce the left ventricular end-systolic anterior wall thickness, significantly reduce the left ventricular end-diastolic anterior wall thickness, significantly reduce the left ventricular end-systolic posterior wall thickness, and significantly reduce the left ventricular end-diastolic posterior wall thickness. Therefore, the Xinsuning preparation can significantly improve cardiac function, and is used in the preparation of drugs for the prevention and treatment of heart failure by improving cardiac function.

[0017] 2. The use of the Xinsuning preparation of the present invention in the preparation of drugs for the prevention and treatment of heart failure, wherein the Xinsuning preparation treatment group of the present invention can significantly reduce myocardial cell edema, reduce myocardial cell inflammatory infiltration, and improve the alignment of myocardial cells compared with the heart failure model group and the control group. Therefore, the Xinsuning preparation can improve myocardial structure and is used in the preparation of drugs for the prevention and treatment of heart failure by improving myocardial structure.

[0018] 3. The Xinsuning preparation of the present invention can also be used as a protective drug for preventing and treating cardiac toxicity induced by doxorubicin. The present invention uses doxorubicin-induced heart failure as a heart failure model. Therefore, the Xinsuning preparation can play a role in the preparation and treatment of doxorubicin-induced heart failure drugs by improving cardiac function and myocardial structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a flowchart of heart failure mouse modeling;

[0020] Figure 2 These are the cardiac ultrasound images of mice in the control group, heart failure model group, and xinsuning treatment group;

[0021] Figure 3 This is a schematic diagram of the cardiac ultrasound characterization values ​​of mice in the control group, heart failure model group, and xinsuning treatment group;

[0022] Figure 4 This is a schematic diagram of the effect of Xinsuning on the pathological morphology of mouse heart tissue after adriamycin-induced cardiomyocyte injury at HE staining and magnification X=4;

[0023] Figure 5 This is a schematic diagram of the effect of Xinsuning on the pathological morphology of mouse heart tissue after adriamycin-induced cardiomyocyte injury, HE staining, at a magnification of X=400;

[0024] Figure 6 Schematic diagram of TUNEL staining of mouse heart tissue after adriamycin-induced cardiomyocyte injury by Xinsuning;

[0025] Figure 7 Schematic diagram of projection electron microscopy of cardiomyocytes. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the implementation modes described below.

[0027] Xinsu Ning preparation is composed of 11 Chinese herbs, including Coptis chinensis, Pinellia tuber, Poria cocos, Citrus aurantium, Dichroa chinensis, Lotus seed core, Sophora flavescens, Artemisia annua, Ginseng, Ophiopogon japonicus, and Licorice, and is mainly used to clear away heat and resolve phlegm, calm the heart and relieve palpitations. Studies have shown that this drug is effective against ventricular premature beats and phlegm-heat disturbance of the heart, and exerts anti-arrhythmic effects through the electrophysiological mechanism of blocking multiple ion channels (NaV1.5, CaV1.2, hERG, etc.) and regulating non-ionic targets (NNI3, CACNA1S, SCN5A, etc.). It is currently the only safe and effective Chinese patent medicine for the treatment of tachyarrhythmias.

[0028] According to the clinical symptoms of arrhythmia and the tongue and pulse, most of them can be classified into the categories of "palpitations" and "palpitations". A few can manifest as chest tightness, chest pain, dizziness and syncope, so they can also be classified into the categories of "chest pain", "vertigo" and "syncope" in traditional Chinese medicine. Heart failure is a group of syndromes with abnormal heart function, clinical manifestations of congestion in the pulmonary and systemic circulation, and insufficient blood perfusion of organs and tissues. Arrhythmia is an abnormality in the frequency, rhythm, origin, conduction velocity or order of cardiac excitation. Therefore, although the Xinsuning preparation has excellent therapeutic advantages in arrhythmia, there has been no relevant research and application in the clinical prevention and treatment of heart failure.

[0029] Although the existing literature reports that ginsenoside Rb1, the main active ingredient of ginseng, can eliminate the delayed depolarization induced by high calcium in rabbit ventricular myocytes, inhibit the increase of intracellular calcium ions, relieve calcium overload, and play an anti-arrhythmic and protective role for myocardial cells. Glycyrrhizic acid, the main active ingredient of licorice, can inhibit the AMPK / mTOR autophagy signaling pathway mediated by high mobility group protein B1 (HMGB1) to reduce doxorubicin-induced cardiotoxicity. Ophiopogon japonicus' main active ingredient Ophiopogon japonicus saponin D protects myocardial cells by reducing doxorubicin-induced ROS accumulation and relieving endoplasmic reticulum stress. Berberine in Coptis chinensis significantly improves doxorubicin-induced myocardial damage by protecting the intestinal mucosal barrier function, reducing endotoxin entry into the blood, relieving myocardial inflammation and reducing myocardial apoptosis. However, the above-mentioned single medicinal flavors do not achieve the effect of preventing and treating heart failure by improving cardiac function. At the same time, Chinese medicine compositions must comply with the theory of Chinese medicine compatibility. Due to the complexity of Chinese medicine ingredients and the interactions between them, the ingredients of Chinese medicine formulas are no longer the sum of the ingredients of the raw materials. Therefore, the efficacy of Chinese medicine formulas is not a simple addition of the efficacy of individual herbs.

[0030] Therefore, the present invention proposes the application of Xinsu Ning preparation in the preparation of drugs for preventing and treating heart failure. Specifically, the application of Xinsu Ning preparation in the preparation of drugs for preventing and treating heart failure induced by adriamycin. Adriamycin (DOX for short), also known as doxorubicin, is one of the better chemotherapy drugs with a wide range of effects. Although adriamycin can be used to treat cancer, adriamycin will produce nephrotoxicity, hepatotoxicity, alopecia, bone marrow suppression, chemotherapy brain and other side effects while treating cancer. The most serious adverse reaction is that adriamycin can induce cardiotoxicity, which is manifested as myocardial cell damage, apoptosis, necrotic cell death, and the occurrence of life-threatening left ventricular dysfunction, arrhythmia and heart failure (heart failure) etc. At present, it is known that there are drugs with potential prevention and treatment of adriamycin cardiotoxicity, in addition to dexamethasone, there are also beta-blockers, angiotensin converting enzyme inhibitors / angiotensin receptor blockers, statins, etc., but the efficacy of these drugs is limited and there is some controversy. Traditionally, dexamethasone is considered to be a prodrug of a metal chelate mixture, which prevents the formation of reactive oxygen by isolating unstable iron ions to reduce myocardial damage, but the applicable diseases are limited. In addition, it also has the risk of secondary malignant tumors. Other cardioprotective agents lack strong evidence-based medicine. The present invention uses adriamycin-induced heart failure as a heart failure model, and the Xinsuning preparation can act in the preparation and treatment of adriamycin-induced heart failure drugs by improving cardiac function and myocardial structure. It can be used as a protective drug for preventing and treating cardiotoxicity induced by adriamycin.

[0031] Xinsu Ning preparation includes: 334 parts of Coptis chinensis, 250 parts of Banxia ternata, 250 parts of Poria cocos, 167 parts of Citrus aurantium, 250 parts of Dichroa chinensis, 42 parts of Lotus Seed Heart, 250 parts of Sophora flavescens, 250 parts of Artemisia annua, 167 parts of Ginseng, 250 parts of Radix Ophiopogonis and 167 parts of Licorice. In the prescription, Coptis chinensis and Banxia ternata are the monarchs, clearing away heat and resolving phlegm; Artemisia annua, Dichroa chinensis, Sophora flavescens and Lotus Seed Heart are the ministers, helping the monarch to clear heat, resolve phlegm and detoxify, clear the heart and calm the mind; Poria cocos and Citrus aurantium are the adjuvants, regulating qi, draining dampness and removing phlegm; Ginseng and Ophiopogonis are also adjuvants, nourishing the heart and restoring the pulse; Licorice is the guiding drug. It conforms to the theory of traditional Chinese medicine and the formula composition rules of monarch, minister, assistant and guiding, and is mainly used to clear heat and resolve phlegm, calm the heart and calm palpitations.

[0032] The Xinsuning preparation is used in the preparation of drugs for preventing and treating heart failure by improving cardiac function. The improvement of cardiac function includes increasing cardiac ejection fraction, improving left ventricular short axis shortening rate, reducing left ventricular end-systolic / diastolic inner diameter, reducing left ventricular end-systolic / diastolic anterior wall thickness and / or reducing left ventricular end-systolic / diastolic posterior wall thickness.

[0033] The Xinsuning preparation is also used in the preparation of drugs for preventing and treating heart failure by improving myocardial structure. Improving myocardial structure refers to improving the changes in myocardial tissue morphology caused by adriamycin. The myocardial tissue morphology includes the degree of myocardial cell edema, the degree of myocardial cell inflammatory infiltration, and the neatness of myocardial cell arrangement.

[0034] The Xinsuning preparation is used in preparing medicines for preventing and treating heart failure by increasing the number of myocardial cell mitochondria.

[0035] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0036] Example 1: Preparation of Xinsuning Capsules

[0037] Composition formula: 334g of Coptis chinensis, 250g of Pinellia ternata, 250g of Poria cocos, 167g of Immature Citrus aurantium, 250g of Dichroa chinensis, 42g of Lotus seed core, 250g of Sophora flavescens, 250g of Artemisia annua, 167g of Ginseng, 250g of Ophiopogon japonicus, and 167g of Licorice.

[0038] The process steps are as follows:

[0039] (1) The raw materials are cleaned, sliced, and dried for later use.

[0040] (2) Take 334g of Coptis chinensis, 167g of Citrus aurantium, 250g of Radix Dichroae, 42g of Pithmum nelumbinis and 250g of Sophora flavescens, add 60% ethanol, and reflux extract twice. For the first time, 8 times the amount of ethanol was added to the raw materials of Coptis chinensis, Citrus aurantium, Radix Dichroae, Pithmum nelumbinis and Sophora flavescens, and reflux extract was performed for 2 hours. For the second time, 6 times the amount of ethanol was added to the raw materials of Coptis chinensis, Citrus aurantium, Radix Dichroae, Pithmum nelumbinis and Sophora flavescens, and reflux extract was performed for 1.5 hours. The extracts were combined and filtered. The filtrate was decompressed at 0.04MPa and 75°C to recover ethanol, and the filtrate was further concentrated to a thick paste with a relative density of 1.36 to 1.38 at 60°C, and dried at 80°C to form a dry paste. The residue was discarded.

[0041] (3) Take 167g of ginseng, 250g of Poria cocos, and 250g of Pinellia ternata, add 70% ethanol, and reflux extract twice. The first time, add 10 times the amount of ethanol of the raw materials of ginseng, Poria cocos, and Pinellia ternata, and reflux extract for 2.5 hours. The second time, add 8 times the amount of ethanol of the raw materials of ginseng, Poria cocos, and Pinellia ternata, and reflux extract for 2 hours. Combine the extracts, filter, and collect the filtrate in another container for later use.

[0042] (4) The residue obtained in step (3) is combined with 250 g of Ophiopogon japonicus, 250 g of Artemisia annua and 167 g of Licorice, and decocted twice with water. The first time, 10 times the amount of water was added to the residue, Ophiopogon japonicus, Artemisia annua and Licorice raw materials, and the second time, 8 times the amount of water was added to the residue, Ophiopogon japonicus, Artemisia annua and Licorice raw materials. Each time, the decoction is combined, filtered, and the filtrate is concentrated to a clear paste with a relative density of 1.05 to 1.06 at 80°C, 95% ethanol is added to make the alcohol concentration reach 70%, mixed, and allowed to stand for 24 hours to precipitate, the supernatant is collected, filtered, the filtrate is combined with the filtrate collected in another device in step (3), and the ethanol is recovered under reduced pressure at 0.04 MPa and 75°C, and the mixture is further concentrated to a thick paste with a relative density of 1.36 to 1.38 at 60°C, and dried at 80°C to obtain a dry paste.

[0043] (5) Combine the two dry pastes obtained in step (2) and step (4), crush into 100-mesh fine powder, add appropriate amount of dextrin, mix well, put into capsules, and prepare 1000 capsules.

[0044] Example 2 Preparation of Xinsu Ning Tablets

[0045] The composition formula of Xinsu Ning Tablets is the same as that of Xinsu Ning Capsules, and the process steps (1) to (4) are also the same as those of Xinsu Ning Capsules, except that:

[0046] (5) The two dry pastes obtained in step (2) and step (4) are combined, crushed into a fine powder of 100 mesh, added with an appropriate amount of dextrin, granulated with 85% ethanol, dried at 60°C, granulated, added with 0.5% magnesium stearate, mixed well, pressed into 1000 tablets, and coated.

[0047] Example 3 Preparation of Xinsu Ning Pills

[0048] The composition formula of Xinsu Ning Pills is the same as that of Xinsu Ning Capsules, and the process steps (1) to (4) are also the same as those of Xinsu Ning Capsules, except that:

[0049] (5) Combine the two dry pastes obtained in step (2) and step (4), grind into fine powder of 100 mesh, add 35g-50g refined honey and appropriate amount of water to make pills for every 100g of powder, dry and make water-honey pills; or add 80-130g refined honey to make small honey pills or large honey pills.

[0050] Example 4 Preparation of Xinsuning Granules

[0051] The composition formula of Xinsu Ning Granules is the same as that of Xinsu Ning Capsules, and the process steps (1) to (4) are also the same as those of Xinsu Ning Capsules, except that:

[0052] (5) The two dry pastes obtained in step (2) and step (4) are combined, and an appropriate amount of sucrose powder is added to form granules, and dried to obtain the granules.

[0053] In the preparation process, auxiliary materials used in pharmacy such as fillers, binders, disintegrants, lubricants, flavoring agents and the like may also be added to oral liquid preparations.

[0054] 1. Take the Xinsuning capsules prepared in Example 1 and carry out the following experimental process.

[0055] 1.1 Experimental Materials

[0056] The experimental instruments and reagents are shown in Table 1.

[0057] Table 1 Main experimental instruments

[0058]

[0059] Xinsuning capsules were purchased from Shaanxi Momede Qixuehe Pharmaceutical Co., Ltd. (approval number: National Medicine Standard Z20050131). Adriamycin, item number: D8740, specification: 500 mg, was purchased from Solebao Company.

[0060] 1.2 Experimental animals

[0061] A total of 30 male C57BL / 6 mice, weighing 20±5g, were kept in the SPF-grade standard animal breeding room of the Experimental Animal Center of Shandong University of Traditional Chinese Medicine. Breeding conditions: temperature controlled at 24±2℃; humidity controlled at 50±10%; artificial light 12h / d; ventilation rate 15 times / h, mice free access to drinking water and food, and the mice used in the experiment were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2021-0011. All animals in the experiments were carried out in strict accordance with the "Guiding Principles for the Care and Use of Laboratory Animals" of Shandong University of Traditional Chinese Medicine. This study has been approved by the Animal Ethics Review Committee of Shandong University of Traditional Chinese Medicine (Approval Number: SDUTCM20230828004).

[0062] 1.3 Drug preparation

[0063] Xinsuning treatment group: Weigh an appropriate amount of Xinsuning capsule powder and add 10 ml of pure water to make 0.075 g kg -1 of aqueous suspension.

[0064] Control group: mice were given normal saline at 10 ml / kg / d.

[0065] Heart failure model group (DOX group) and xinsuning treatment group (DOX+XSN group): The modeling drug was prepared at 3 mg / kg, 15 mg of doxorubicin was dissolved in 50 ml of normal saline, and immediately divided into 5 centrifuge tubes and stored at 4°C in the dark for 1 month. The mice were intraperitoneally injected with the modeling drug at 3 mg / kg.

[0066] 1.4 Experimental Procedure

[0067] (1) Grouping: control group (CON group), heart failure model group (DOX group), and XSN treatment group (DOX+XSN group).

[0068] (2) Heart failure modeling: Male C57BL / 6 mice were injected intraperitoneally with doxorubicin for 5 consecutive days in the heart failure model group (DOX group) and the XSN treatment group (DOX+XSN group). The control group (CON group) was injected intraperitoneally with an equal amount of normal saline.

[0069] (3) Administration: The DOX+XSN group was given drugs by intragastric administration for 14 consecutive days, with a dosage of 10 mL kg -1 ; The control group (CON group) was intragastrically administered with an equal volume of normal saline.

[0070] (4) Experimental flow chart, see Figure 1 .

[0071] 1.5 Experimental methods

[0072] (1) Echocardiography in mice

[0073] Preparation of coupling agent: After completing the 14-day modeling and intervention experiment, each group of mice was subjected to ultrasound examination. The coupling agent was placed in a 50 mL EP tube and centrifuged at 2000 rpm for 5 minutes to remove the excess steam in the coupling agent. Ultrasonic examination: Anesthesia was induced using isoflurane (concentration 5%, ventilation volume 1 L min -1 ) After that, the mouse was fixed on the ultrasound operating table in a supine position and anesthetized using a mask (2% concentration, ventilation volume 0.4 L·min -1 ), turn on the thermostat to maintain the body temperature of the mouse at around 37°C to ensure that the animal is in the best physiological state. Use a cotton swab to evenly apply the depilatory cream to the mouse's chest. After waiting for 2-3 minutes, use moistened absorbent paper to wipe off the hair and fully expose the mouse's lower chest skin. Apply the coupling agent evenly to the mouse's chest skin, then slowly bring the ultrasound probe close to the heart and observe the imaging results. Place the probe horizontally at the position of the sternum, use B-Mode to locate the long axis of the left ventricle, and then rotate the probe clockwise 30°-45° according to the determined position to locate the short axis of the left ventricle. Use M-Mode to observe and record the cyclic contraction and relaxation of the ventricular muscle.

[0074] (2) Hematoxylin-eosin staining (HE staining)

[0075] In order to observe the cell structure, cytoplasm and nuclear morphology of myocardial tissue, HE staining was performed. Hematoxylin is alkaline, which makes the chromatin in the nucleus and the ribosomes in the cytoplasm blue-purple; eosin is an acidic dye, which makes the cytoplasm and extracellular matrix components red. The steps are as follows: 1. Sampling: After completely removing the mouse heart, place the mouse heart in the direction of the ultrasonic long axis section and put the mouse heart into the 4% paraformaldehyde prepared in advance. 2. Dehydration and embedding: After the mouse heart tissue block is fixed in 4% paraformaldehyde for 2 hours, it is placed in a dehydrator for dehydration. After dehydration, it is embedded in paraffin and the embedded wax block is placed in a 4°C refrigerator for standby use. 3. Staining: 1) Take the dewaxed tissue specimen, stain with hematoxylin for 10 minutes, put it in pure water, and wash it; 2) 1% hydrochloric acid and ethanol for 1 second each, wash it, and immediately put it in warm water to turn blue (20 minutes), which is the weathering process; 3) Eosin for 2 seconds, put it in tap water and wash it clean. Observe the staining results under the microscope and you can see that the cell nucleus is blue, and the cytoplasm, muscle fibers, collagen fibers and red blood cells are different shades of red; 4) Transparent: rinse until the water is clean, blow dry the slide with a hair dryer, and stain according to the process of anhydrous ethanol I→anhydrous ethanol II→xylene I→xylene II→xylene III, each for 1 minute; 5) Sealing: wipe the water around the slide tissue clean, drop a drop of sealing agent, and cover the slide.

[0076] (3) TUNEL staining

[0077] The embedded paraffin specimens were sliced ​​with a paraffin slicer, dried at 60°C for 2 hours, dewaxed, dehydrated with graded alcohol, and then stained according to the instructions of the kit. The slices were transparentized with graded alcohol and xylene and sealed, and photographed with an inverted fluorescence microscope.

[0078] 1.6 Statistical analysis

[0079] The experimental data were analyzed using SPSS26.0 software, and the statistical graphs were drawn using GraphPadPrism8.30 software. The measurement data were expressed as mean ± standard deviation (X ± SEM). One-way ANOVA was used to compare repeated measurement data of multiple groups, and LSD-t test was used to compare data between two groups. P < 0.05 was considered statistically significant. All statistical results were tested with a test statistic α = 0.05 as the significance test standard. *P < 0.05, **P < 0.01, ***P < 0.001.

[0080] 1.7 Experimental Results

[0081] 1.7.1 Xinsuning preparation can significantly improve the cardiac function of heart failure mice

[0082] See also Figure 2 The cardiac ultrasound images of mice in the control group (CON group), heart failure model group (DOX group) and XSN treatment group (DOX+XSN group); see Figure 3 Schematic diagram of the cardiac ultrasound characterization values ​​of mice in the control group (CON group), heart failure model group (DOX group) and xinsuning treatment group (DOX+XSN group); Figure 3 A: Statistics of left ventricular ejection fraction of mice. The ejection fraction of the heart failure model group (DOX group) was significantly lower than that of the control group (CON group). The ejection fraction of the xinsuning treatment group (DOX+XSN group) was significantly improved compared with the heart failure model group (DOX group) and the control group (CON group). Figure 3 B: Statistics of the short-axis shortening rate of the left ventricle in mice. The short-axis shortening rate of the heart failure model group (DOX group) was significantly lower than that of the control group (CON group). Compared with the heart failure model group (DOX group) and the control group (CON group), the short-axis shortening rate of the xinsuning treatment group (DOX+XSN group) was significantly improved. Figure 3 C: Statistics of the left ventricular end-systolic diameter of mice. The left ventricular end-systolic diameter of the heart failure model group (DOX group) was significantly increased compared with that of the control group (CON group). Compared with the heart failure model group (DOX group) and the control group (CON group), the xinsuning treatment group (DOX+XSN group) can significantly reduce the left ventricular end-systolic diameter. Figure 3D: Statistics of the left ventricular end-diastolic diameter of mice. The left ventricular end-diastolic diameter of the heart failure model group (DOX group) was significantly increased compared with the control group (CON group). Compared with the heart failure model group (DOX group) and the control group (CON group), the xinsuning treatment group (DOX+XSN group) can significantly reduce the left ventricular end-diastolic diameter. Figure 3 E: Statistics of the anterior wall thickness of the left ventricle at the end of systole in mice showed that the anterior wall thickness of the heart failure model group (DOX group) was significantly thinner than that of the control group (CON group). Compared with the heart failure model group (DOX group) and the control group (CON group), the xinsuning treatment group (DOX+XSN group) could improve the thinning of the anterior wall thickness of the left ventricle at the end of systole. Figure 3 F: Statistics of the left ventricular anterior wall thickness at the end of diastole in mice showed that the anterior wall thickness in the heart failure model group (DOX group) was significantly thinner than that in the control group (CON group). Compared with the heart failure model group (DOX group) and the control group (CON group), the xinsuning treatment group (DOX+XSN group) could improve the thinning of the left ventricular anterior wall thickness at the end of diastole. Figure 3 G: Statistics of the thickness of the posterior wall of the left ventricle at the end of systole in mice showed that the anterior wall thickness of the heart failure model group (DOX group) was significantly thinner than that of the control group (CON group). Compared with the heart failure model group (DOX group) and the control group (CON group), the xinsuning treatment group (DOX+XSN group) could improve the thinning of the posterior wall thickness of the left ventricle at the end of systole. Figure 3 H: Statistics of the left ventricular posterior wall thickness at the end of diastole in mice showed that the anterior wall thickness of the heart failure model group (DOX group) was significantly thinner than that of the control group (CON group). Compared with the heart failure model group (DOX group) and the control group (CON group), the xinsuning treatment group (DOX+XSN group) could improve the thinning of the left ventricular posterior wall thickness at the end of diastole.

[0083] See also Figure 7 The results of projection electron microscopy of mouse cardiomyocytes showed that mitochondria in the myocardial tissue of the heart failure model group (DOX group) showed autophagy, while the group treated with XSN (DOX+XSN group) did not block mitochondrial autophagy and greatly increased the number of mitochondria. This shows that XSN preparations can improve cardiac function by increasing the number of mitochondria in cardiomyocytes.

[0084] 1.7.2 Xinsuning can improve the myocardial structure of mice with heart failure

[0085] See also Figure 4 and Figure 5The myocardium of mice in the control group (CON group) was tightly arranged, the thickness of myofilaments was uniform, the nuclei were clearly distributed in the center, the cytoplasm was uniform in color, the intercellular spaces were normal, and there was no inflammatory cell infiltration; the myocardium of mice in the heart failure model group (DOX group) was disordered, the myocardium was staggered, accompanied by inflammatory cell infiltration, myocardial cell coagulation necrosis, and the cytoplasm color was different in depth. The xinsuning treatment group (DOX+XSN group) significantly reduced myocardial cell edema, reduced inflammatory cell infiltration, myocardial cells were neatly arranged, and the cytoplasm was uniformly colored. It can be seen that the xinsuning preparation can improve the changes in myocardial tissue morphology of mice induced by doxorubicin.

[0086] 1.7.3 Xinsuning can significantly improve myocardial cell apoptosis in heart failure mice

[0087] See also Figure 6 ,Compared with the control group (CON group), the number of myocardial apoptotic cells in the mice in the heart failure model group (DOX group) was more than that in the xinsuning treatment group (DOX+XSN group), indicating that the xinsuning preparation can improve the apoptosis of myocardial cells in mice with heart failure.

[0088] 2. Comparative test

[0089] To further demonstrate the role of Xinsuning preparation in the preparation of drugs for the prevention and treatment of heart failure, the following experiment was conducted.

[0090] 2.1. Formula:

[0091] Xinsuning group: Coptis chinensis 334g, Pinellia ternata 250g, Poria 250g, Citrus aurantium 167g, Dichroa 250g, Lotus seed heart 42g, Sophora flavescens 250g, Artemisia annua 250g, Ginseng 167g, Ophiopogon japonicus 250g, and Licorice 167g

[0092] Comparative Example 1 group: The difference from the Xinsuning group is that it does not contain Coptis chinensis.

[0093] Comparative Example 2 Group: The difference from the Xinsu Ning Group is that it does not contain Pinellia ternata, Radix Dichroae and Lotus Seed Heart.

[0094] Comparative Example 3 Group: The difference from the Xinsu Ning Group is that Coptis chinensis and Pinellia ternata are replaced with Honeysuckle and Rhizoma Anemarrhenae.

[0095] Comparative Example 4 group: The difference from the Xinsuning group is that Poria cocos and Sophora flavescens are replaced by Atractylodes macrocephala and Scutellaria baicalensis.

[0096] 2.2 Experimental Animals

[0097] Same as section 1.2.

[0098] 2.3 Drug configuration

[0099] According to the formulation in 2.1, 10 ml of pure water was added to the Xinsuning group and the control group to prepare 0.075 g kg -1of aqueous suspension.

[0100] Control group (CON group): mice were given normal saline at 10 ml / kg / d.

[0101] The model group (DOX group), xinsuning group and control group were all prepared with modeling drugs at 3 mg / kg. 15 mg of doxorubicin was dissolved in 50 ml of normal saline, and the mice were intraperitoneally injected with modeling drugs at 3 mg / kg.

[0102] 2.4 Experimental Process

[0103] (1) Grouping: control group (CON group), model group (DOX group), xinsuning group, comparative example group 1, comparative example group 2, comparative example group 3 and comparative example group 4.

[0104] (2) Heart failure modeling: Male C57BL / 6 mice were intraperitoneally injected with doxorubicin for 5 consecutive days. The control group mice were intraperitoneally injected with an equal amount of normal saline.

[0105] (4) Administration: Xinsuning group and control group: continuous intragastric administration for 14 days, with a dosage of 10 mL kg -1 , Control group: gavage with equal amount of normal saline.

[0106] 2.5. Echocardiography in mice

[0107] As shown in Table 2, compared with the model group (DOX group) and the control group (DOX+XSN-Z group), the Xinsuning group can significantly improve the ejection fraction of the mouse heart; compared with the control groups 1 to 4, the Xinsuning group has a better effect.

[0108] Table 2 Ejection fraction statistics of cardiac detection

[0109]

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Xinsuning preparation in the preparation of drugs for the prevention and treatment of heart failure.

2. The use of the Xinsuning preparation according to claim 1 in the preparation of drugs for preventing and treating heart failure, characterized in that: The Xinsuning preparation is used in preparing medicines for preventing and treating heart failure by improving heart function.

3. The use of the Xinsuning preparation according to claim 2 in the preparation of drugs for preventing and treating heart failure, characterized in that: The improvement of cardiac function includes increasing cardiac ejection fraction, improving left ventricular minor axis shortening rate, reducing left ventricular end-systolic / diastolic inner diameter, reducing left ventricular end-systolic / diastolic anterior wall thickness and / or reducing left ventricular end-systolic / diastolic posterior wall thickness.

4. The use of the Xinsuning preparation according to claim 1 in the preparation of drugs for preventing and treating heart failure, characterized in that: The Xinsuning preparation is used in preparing medicines for preventing and treating heart failure by increasing the number of myocardial cell mitochondria.

5. The use of the Xinsuning preparation according to claim 1 in the preparation of drugs for preventing and treating heart failure, characterized in that: The Xinsuning preparation is used in preparing drugs for preventing and treating heart failure by improving myocardial structure.

6. Use of the Xinsuning preparation according to claim 5 in preparing drugs for preventing and treating heart failure, characterized in that: The improvement of myocardial structure refers to improving the changes induced by adriamycin on myocardial tissue morphology.

7. Use of the Xinsuning preparation according to claim 6 in preparing drugs for preventing and treating heart failure, characterized in that: The myocardial tissue morphology includes the degree of myocardial cell edema, the degree of myocardial cell inflammatory infiltration, and the degree of myocardial cell arrangement.

8. Use of the Xinsuning preparation according to claim 1 in preparing drugs for preventing and treating heart failure, characterized in that: The Xinsuning preparation is used in preparing drugs for preventing and treating adriamycin-induced heart failure.

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