Traditional Chinese medicine composition for treating heart failure

Abstract

The invention discloses a traditional Chinese medicine composition for treating heart failure, and particularly relates to the field of traditional Chinese medicine compositions. Comprising the following raw materials in parts by weight: 160 parts of red ginseng, 160 parts of dogwood and 80 parts of salvia miltiorrhiza. The traditional Chinese medicine composition in the scheme achieves the purpose of treating heart failure by regulating and controlling a Hippo-YAP pathway. By adopting the technical scheme, a new method is provided for treating the heart failure at present, and the traditional Chinese medicine composition has the advantage of low cost, is beneficial to reducing the myocardial fibrosis degree of a heart failure mouse in an experiment, and is beneficial to improving the cardiac function of a patient and improving the life quality in a clinical test.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine compositions, and particularly to a traditional Chinese medicine composition for treating heart failure. Background Art

[0002] Chronic heart failure (CHF) usually develops from various cardiovascular diseases and is a clinical syndrome mainly characterized by symptoms such as dyspnea, lower limb edema, and palpitations, usually accompanied by symptoms such as jugular venous hypertension and limb swelling. According to clinical statistics, in 2022, there were nearly 8.9 million CHF patients in China, and the mortality rate was about 4.1%. Heart failure brings a heavy medical burden to society due to its high prevalence, low survival rate, and profound impact on the quality of life of patients. After the occurrence of heart failure, it usually leads to pathological remodeling of the heart, among which myocardial fibrosis is the most important morphological change in cardiac remodeling and is also an important marker of heart failure. Currently, the methods for treating heart failure include drug treatment, resynchronization therapy, and human implantable cardioverter-defibrillators, etc. However, these methods still have problems such as high cost and difficulty in controlling heart failure. Therefore, it is of great significance to find new CHF treatment methods clinically. Summary of the Invention

[0003] The present invention aims to provide a traditional Chinese medicine composition for treating heart failure, providing a new method for the difficult treatment of existing heart failure and having the advantage of low cost.

[0004] To achieve the above object, a technical solution provided by the present invention is as follows: A traditional Chinese medicine composition for treating heart failure, comprising raw materials in the following weight components: 160 parts of red ginseng, 160 parts of cornel, and 80 parts of salvia miltiorrhiza.

[0005] Another technical solution provided by the present invention is as follows: Application of a traditional Chinese medicine composition for treating heart failure in regulating the Hippo-YAP pathway.

[0006] Compared with the prior art, the beneficial effects of this solution are:

[0007] 1. This traditional Chinese medicine composition activates the Hippo-YAP pathway, resulting in the degradation of YAP in the cytoplasm, and the reduced expression of YAP reduces myocardial fibrosis in mice.

[0008] 2. This traditional Chinese medicine composition can improve the cardiac function of CHF mice, reduce the degree of myocardial fibrosis, and reduce the expression of fibrosis-related proteins and YAP in myocardial tissues.

[0009] 3. This traditional Chinese medicine combination can improve the cardiac function of chronic heart failure patients and improve the quality of life. Description of the Drawings

[0010] Figure 1 These are the pathological change diagrams of the heart tissues of each group of mice (H&E);

[0011] Figure 2 These are the pathological change diagrams of the heart tissues of each group of mice (MASSON staining);

[0012] Figure 3 These are the TUNEL staining result diagrams of the myocardial tissues of each group of mice;

[0013] Figure 4 These are the diagrams showing the effects of the traditional Chinese medicine composition of Example 1 on the positive expressions of Col-Ⅰ and Col-Ⅲ in the hearts of each group of mice (immunofluorescence, ×400);

[0014] Figure 5 These are the diagrams showing the effects of the traditional Chinese medicine composition of Example 1 on the positive expressions of p-YAP and p-LATS in the hearts of each group of mice (immunofluorescence, ×400);

[0015] Figure 6 These are the diagrams showing the effects of the traditional Chinese medicine composition of Example 1 on the protein expressions of YAP, p-YAP, LATS1, p-LATS1, Col-Ⅰ, Col-Ⅲ, and α-SMA in the myocardial tissues of each group of mice;

[0016] Figure 7 These are the preparation process diagrams of the traditional Chinese medicine composition of Example 1. Detailed implementation manners

[0017] The present invention will be further described in detail below through specific implementation manners:

[0018] Example 1

[0019] A traditional Chinese medicine composition for treating heart failure, comprising raw materials in the following weight components: 160 parts of red ginseng, 160 parts of cornel, and 80 parts of salvia miltiorrhiza.

[0020] The preparation process of the traditional Chinese medicine composition of this example is as follows:

[0021] As Figure 7 shown, each of the above raw materials is pulverized and then weighed, the corresponding weight components are weighed and mixed, and the mixed composition is filled into capsules and then packaged in bottles.

[0022] Example 2

[0023] Application of the traditional Chinese medicine composition of Example 1 in regulating the Hippo-YAP pathway.

[0024] The traditional Chinese medicine composition of Example 1 is subjected to the following animal experiments and subsequent clinical trials. The materials and methods are as follows:

[0025] Animal selection:

[0026] Sixty 8-week-old SPF-grade male C57BL / 6 mice were used in the experiment. They were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., with the license number SCXK (Sichuan) 2020-0030, and the animal certificate numbers 430727221101891171 and 430727221101836883. They were raised in the SPF-grade laboratory of the Experimental Animal Center of Southwest Medical University. The experimental protocol was approved by the Experimental Animal Ethics Committee of Southwest Medical University, approval number: SWMU20240615.

[0027] Drug selection:

[0028] Isoproterenol hydrochloride (MedChemExpress, batch number: HY-B0468 / CS-2582); capsules prepared from the traditional Chinese medicine composition of Example 1 (specification 0.4 g / capsule), provided by the Preparation Room of the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University.

[0029] Reagent selection:

[0030] Omni-Easy TM One-step PAGE gel rapid preparation kit (product number: PG212) was purchased from Ya Mei Company; two-color standard pre-stained protein (Marker product number: 1610374) was purchased from Bio-rad Company; BCA protein quantification kit (product number: ZJ101) was purchased from Ya Mei Company; RIPA protein lysate (product number: R0010) was purchased from solarbio company, and YAP, p-YAP, LATS1, p-LATS1, α-SMA, Col-I, Col-Ⅲ, GAPDH antibodies (product numbers: AF6328, AF3328, AF7669, AF1032, AF7001, AF5457, AF7021) were purchased from Affinity Company; double-sensitive ECL chemiluminescence reagent (product number: KF005) was purchased from Affinity Company; DAB kit (product number: G2370) was purchased from Solarbio Company; horseradish peroxidase-labeled secondary antibody (product number: S0011) was purchased from Affinity Company; hematoxylin-eosin staining agent, Masson trichrome staining kit, mouse BNP ELISA kit, mouse IL-6 ELISA kit (product numbers: G1120, G1340, SEKR-0058, SEKR-0005) were purchased from Solarbio Company; cover slips and glass slides (product numbers: 10212424C, 10211818C) were purchased from Shitai Company; neutral balsam (product number: G8590) was purchased from Solarbio Company; PCR primers were purchased from Shanghai Sangon Biotech Co., Ltd.; RNA reverse transcription kit (product number: DBI-2220) was purchased from Bioscience. The one-step TUNEL apoptosis detection kit (product number: CX107S) was purchased from YaMei Biotechnology Co., Ltd.

[0031] Instrument selection:

[0032] R530 small animal anesthesia machine (Shenzhen Rewod Life Science Co., Ltd.); R415 small animal ventilator (Shenzhen Rewod Life Science Co., Ltd.); CKX53 Leica optical microscope (Olympus Corporation, Japan); NIKON ECLIPSE TI-SR inverted microscope (Nikon Corporation, Japan); Vevo 3100 high-resolution small animal ultrasound imaging system (FUJIFILM VisualSonics, Canada); RM2245 paraffin slicer (Leica Microsystems, Germany); EG1150 tissue embedding machine (Leica Microsystems, Germany); paraffin embedding cold table (Leica Microsystems, Germany); Ynergy 2 multi-functional microplate reader (BioTek Instruments, Inc., USA); LightCycler 480II Real-time PCR instrument (Roche Diagnostics, USA); ChemiScope 6200 chemiluminescence imaging system (Clinx Science Instruments Co., Ltd., China); vertical electrophoresis equipment (Bio-rad Laboratories, Inc., USA).

[0033] Experimental method:

[0034] Model establishment and model evaluation criteria:

[0035] In this experiment, an ISO-induced mouse CHF model was used. The mice were randomly divided into the following groups: normal control group, model group, low-dose group of the traditional Chinese medicine composition capsule of Example 1 at 1.56 g / kg, medium-dose group of the traditional Chinese medicine composition capsule of Example 1 at 3.12 g / kg, high-dose group of the traditional Chinese medicine composition capsule of Example 1 at 6.24 g / kg. The number of mice in each group was n = 6. The mice in the model group were injected subcutaneously at multiple points on the back with ISO at a dose of 5 mg·kg -1 , and the injection concentration was 1.67 mg·mL -1 , and the mice in the normal group were injected subcutaneously at multiple points on the back with an equal amount of normal saline for 14 consecutive days, and then fed normally for 14 days. The traditional Chinese medicine composition capsule groups were gavaged to the mice according to the dose. When the value of the left ventricular end-diastolic diameter (LVEDD) of the mice was less than 50%, it was proved that the model was successfully established.

[0036] Cardiac function detection:

[0037] After anesthetizing and fixing the mice on the mouse board, their hair was removed. Using a small animal ultrasound system, an M-mode ultrasound probe was placed at a 10°-30° angle to the midline of the sternum on the left chest of the mice. The left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were continuously measured for 3 cardiac cycles in the left ventricular long-axis section. The measurements were taken 3 times continuously and averaged. The left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated.

[0038] Specimen collection:

[0039] After the cardiac function was detected, the mice were anesthetized with 1% sodium pentobarbital, the abdominal aorta of the mice was exposed, and more than 0.5 mL of blood was collected. The collected blood samples were left to stand at room temperature for 2 h and then centrifuged at 3000 r / min. After centrifugation, the supernatant was stored in a -80 °C refrigerator for later use. After collecting blood from the abdominal aorta, the ribs were cut open to expose the thoracic cavity. The heart was quickly dissected after repeated washing with normal saline. The surrounding connective tissues and blood vessels were cut off, and after blotting with filter paper, the heart mass was weighed using an electronic balance. After weighing, the heart tissue was fixed with 4% paraformaldehyde solution, the tissue was placed in a cryopreservation tube and stored in a -80 °C refrigerator for a long time.

[0040] Determination of serum BNP and IL-6 levels by ELISA method:

[0041] The collected mouse blood was centrifuged multiple times and the supernatant was retained. Strictly according to the instructions of the ELISA kit, the ELISA method was used to determine the contents of serum BNP and IL-6.

[0042] Determination of the expression of Col-Ⅰ, Col-Ⅲ, p-YAP and p-LATS in mouse myocardial tissue by immunofluorescence method:

[0043] After the heart tissue was fixed with 4% paraformaldehyde for 24 h, it was embedded and sectioned (4 μm thick). Then, the endogenous peroxidase activity was blocked with 3% hydrogen peroxide at room temperature for 10 minutes. Then the sections were blocked with 5% bovine serum albumin for one hour. After blocking, the sections were incubated in a mixture of primary antibodies (i.e., Col-Ⅰ + Col-Ⅲ and p-YAP + p-LATS) at a dilution of 1:100 overnight at 4 °C. After incubation with the primary antibody, the sections were incubated with the secondary antibody for 60 min, then rinsed with PBS, developed with DAB, dehydrated and sealed. Photographs were taken using an inverted fluorescence microscope (400×), and 6 fields of view were randomly selected from each section.

[0044] Determination of the mortality rate of cardiomyocytes in mouse myocardial tissue by TUNEL staining:

[0045] After fixing the heart tissue with 4% paraformaldehyde for 24 h, embed it and section it (4 μm thick). Then cover the tissue with the reaction solution and incubate at 37 °C for 2 h. Cover the tissue with BSA and block it at room temperature for 30 min. Incubate with the primary antibody and incubate overnight at 4 °C. After washing with PBS, incubate with the secondary antibody (dilution ratio 1:100) at room temperature in the dark for 60 min. After washing with PBS, incubate with DAPI staining solution and incubate at room temperature in the dark for 10 min. Wash the slides again and mount them with an anti-fluorescence quenching mounting medium. Take pictures with an inverted fluorescence microscope. Randomly select 6 fields of view for each section and use Image-Pro Plus 6.0 software to count the number of positive cells and the total number of nucleated cells in each field of view. Positive cell rate (%) = (number of positive cells / total number of nucleated cells) × 100%.

[0046] Pathological histological observation:

[0047] After fixing the heart tissue with 4% paraformaldehyde for 24 h, embed it and section it (4 μm thick), and operate according to the kit instructions. After routine dewaxing and rehydration, drop the iron hematoxylin mixture on the tissue, stain for 5 min, wash with running water, drop the ponceau staining solution, let it stand and wash with water, then drop the phosphomolybdic acid staining solution. After differentiation, shake off the phosphomolybdic acid staining solution, drop the toluidine blue and stain for 2 min. Wash with running water and then wash the section with 1% glacial acetic acid. Place the section in glacial acetic acid for 1 min, dehydrate with 100% ethanol for 1 min, air dry and seal with neutral gum. Observe under the microscope and save the image.

[0048] RT-qPCR:

[0049] Detect the expression of YAP and myocardial fibrosis-related factor mRNAs in mouse myocardial tissue, and extract total RNA from the heart tissue according to the kit instructions. Use a fluorescence quantitative PCR instrument for routine melting curve analysis and determination of the Ct value after reverse transcription. GAPDH is used as an internal reference gene. The relative expression level of mRNA uses the 2- ΔΔ Ct method. The primer sequences of YAP, LATS1, Col-Ⅰ, and Col-Ⅲ are provided in Table 1.

[0050] Table 1 Primer sequences

[0051]

[0052]

[0053] Western blot:

[0054] After grinding the tissue, proteins in the myocardial tissue were extracted, and a BCA kit was used to test the protein concentration of the samples. After electrophoresis, membrane transfer, and blocking, the primary antibodies were diluted with 1×PBST in the following ratios: YAP (1:1000), p-YAP (1:1000), LATS1 (1:1000), p-LATS1 (1:1000), Col-Ⅰ (1:1000), Col-Ⅲ (1:1000), α-SMA (1:1000), GAPDH (1:10000). After dilution, the PVDF membrane was incubated with the primary antibodies overnight. After incubation, it was washed 3 times with 1×PBST for 10 minutes each time. The HRP secondary antibody [goat anti-rabbit IgG (H+L) secondary antibody HRP (1:10000)] was diluted with 1×PBST, and then the secondary antibody and the membrane were incubated together on a shaker at room temperature for 60 minutes. After incubation, it was washed 3 times with 1×PBST for 10 minutes each time. Subsequently, the membrane was incubated in the ECL chemiluminescent solution and imaged in a gel system. After imaging, the relative protein expression levels were analyzed and calculated using Image Lab software, with GAPDH as the internal reference.

[0055] Statistical methods:

[0056] The software GraphPad Prism8 was used for statistical analysis of the data. Measurement data were expressed as mean ± standard deviation (s), and one-way analysis of variance (One-Way ANOVA) was used. P<0.05 and P<0.01 indicated that there were statistically significant differences between the two groups.

[0057] Experimental results:

[0058] Effect of the traditional Chinese medicine composition capsules on cardiac function in heart failure model mice

[0059] Echocardiogram parameters showed that after the drug intervention, the anterior wall of the ventricle in the model group was thinner than that in the normal control group, the posterior wall was compensatorily hypertrophied, the movement of the anterior wall was weakened, and the LVEF and LVFS indexes were decreased, while the LVEDD and LVESD indexes were increased (P<0.01). Compared with the model group, the LVEF and LVFS in the low-dose traditional Chinese medicine composition capsule, medium-dose traditional Chinese medicine composition capsule, and high-dose traditional Chinese medicine composition capsule administration groups were increased (P<0.01), and the LVEDD and LVESD indexes were significantly decreased (P<0.01). The results are shown in Table 2 below.

[0060] Table 2 Effect of the traditional Chinese medicine composition capsules on cardiac function in heart failure model mice (x±s; n = 6)

[0061]

[0062]

[0063] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0064] Effect of the traditional Chinese medicine composition capsule on the pathological structure of the heart tissue of heart failure model mice

[0065] In the normal control group of mice, H&E staining showed that the myocardial structure and morphology were normal, the myocardial bundles were neatly arranged without swelling or rupture. In the model group, the myocardial cells were hypertrophied and arranged disorderly. In the low-dose traditional Chinese medicine composition capsule group, the middle-dose traditional Chinese medicine composition capsule group, and the high-dose traditional Chinese medicine composition capsule group, the disordered arrangement of myocardial cells was significantly improved. Masson staining showed that there was no blue staining of collagen fibers in the myocardial interstitium of the normal control group. In the model group, a large number of blue collagen fibers connected in a network were visible in the myocardial interstitium and around blood vessels. The blue collagen fibers in the myocardial interstitium of the low, middle, and high-dose traditional Chinese medicine composition capsule groups were reduced compared with the model group. The results are shown in Figure 1 and Figure 2 , where A: normal group, B: model group, C: low-dose traditional Chinese medicine composition capsule group, D: middle-dose traditional Chinese medicine composition capsule group, E: high-dose traditional Chinese medicine composition capsule group. Scale bar = 100 μm.

[0066] Effect of the traditional Chinese medicine composition capsule on apoptosis of myocardial cells in heart failure model mice

[0067] Compared with the control group, the number of TUNEL-stained positive cells in the myocardial tissue of mice in the model group increased (P<0.01). Compared with the model group, the number of TUNEL-stained positive cells in the myocardial tissue of each administration group decreased (P<0.01). The results are shown in Figure 3 and Table 3 below, where A: normal group, B: model group, C: low-dose traditional Chinese medicine composition capsule group, D: middle-dose traditional Chinese medicine composition capsule group, E: high-dose traditional Chinese medicine composition capsule group. Scale bar = 100 μm.

[0068] Table 3 Effect of the traditional Chinese medicine composition capsule on the apoptosis rate of myocardial cells in heart failure model mice (x±s; n = 6)

[0069]

[0070]

[0071] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0072] Effect of the traditional Chinese medicine composition capsule on the contents of serum BNP and IL-6 in heart failure model mice

[0073] Compared with the normal control group, the levels of BNP and IL-6 in the model group of mice increased (P<0.01); compared with the model group, the levels of BNP and IL-6 in each drug administration group decreased (P<0.01). The results are shown in Table 4 below.

[0074] Table 4 Effects of the traditional Chinese medicine composition capsules on serum BNP and IL-6 in heart failure model mice (x±s; n = 6)

[0075]

[0076]

[0077] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0078] Effects of the traditional Chinese medicine composition capsules on the expressions of Col-Ⅰ, Col-Ⅲ, p-YAP and p-LATS in the myocardial tissues of heart failure model mice

[0079] The results of immunofluorescence staining showed that compared with the normal control group, the expressions of Col-Ⅰ and Col-Ⅲ in the model group increased (P<0.01), and the expressions of p-YAP and p-LATS decreased (P<0.01); compared with the model group, the expressions of Col-Ⅰ and Col-Ⅲ in the traditional Chinese medicine composition capsule administration group gradually decreased according to the administration dose (P<0.01), and the expressions of p-YAP and p-LATS gradually increased according to the administration dose (P 0.01). The results are shown in Figure 4 、 Figure 5 Table 5 and Figure 4 and Figure 5 In, A: normal group B: model group C: low-dose traditional Chinese medicine composition capsule group D: medium-dose traditional Chinese medicine composition capsule group E: high-dose traditional Chinese medicine composition capsule group. Scale = 100μm.

[0080] Table 5 Effects of the traditional Chinese medicine composition capsules on the positive expressions of Col-Ⅰ, Col-Ⅲ, p-YAP and p-LATS in the hearts of mice in each group (x±s; n = 6)

[0081]

[0082]

[0083] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0084] Effect of Traditional Chinese Medicine Composition Capsule on the Expression of YAP, Col-Ⅰ, Col-Ⅲ, LATS1 and α-SMA mRNA in Myocardial Tissue of Heart Failure Model Mice

[0085] Compared with the normal control group, the mRNA expressions of YAP, Col-Ⅰ, Col-Ⅲ, LATS1 and α-SMA in the model group were up-regulated (P<0.01); compared with the model group, the mRNA expressions of YAP, Col-Ⅰ, Col-Ⅲ, LATS1 and α-SMA in the Traditional Chinese Medicine Composition Capsule administration group were down-regulated (P<0.01). The results are shown in Table 6 below.

[0086] Table 6 Effect of Traditional Chinese Medicine Composition Capsule on the Expression of YAP, Col-Ⅰ, Col-Ⅲ, α-SMA and LATS1 mRNA in Myocardial Tissue of Heart Failure Model Mice (x±s; n = 3)

[0087]

[0088]

[0089] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0090] Effect of Traditional Chinese Medicine Composition Capsule on the Protein Expressions of YAP, p-YAP, LATS1, p-LATS1, Col-Ⅰ, Col-Ⅲ and α-SMA in Myocardial Tissue of Heart Failure Model Mice

[0091] Compared with the normal control group, the protein expressions of Col-Ⅰ, Col-Ⅲ, YAP, LATS1 and α-SMA in the model group were up-regulated (P<0.01), while p-YAP and p-LATS1 were down-regulated (P<0.01); compared with the model group, the protein expressions of Col-Ⅰ, Col-Ⅲ, YAP, LATS1 and α-SMA in the Traditional Chinese Medicine Composition Capsule administration group were down-regulated, while the expressions of p-YAP and p-LATS1 increased (P<0.01). The results are shown in Figure 6 and Table 7 below.

[0092] Table 7 Effect of Traditional Chinese Medicine Composition Capsule on the Protein Expressions of YAP, p-YAP, LATS1, p-LATS1, Col-Ⅰ, Col-Ⅲ and α-SMA in Myocardial Tissue of Heart Failure Model Mice (x±s; n = 3)

[0093]

[0094]

[0095] Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01.

[0096] Technical principle and effect: Due to its high readmission rate, morbidity and mortality, CHF has occupied a large amount of clinical medical resources in recent years and has always been a hot topic and difficult problem in the field of cardiovascular research. Myocardial fibrosis is an important morphological change in CHF. Its pathological process is mainly manifested as abnormal increase of cardiac fibroblasts, excessive deposition of extracellular matrix of cardiomyocytes, imbalance of the ratio of type I and type III collagen, resulting in scar formation and restriction of injury, which will lead to further cardiac lesions and ultimately heart failure. Therefore, it is of great significance to explore the molecular targets affecting myocardial fibrosis during the process of CHF.

[0097] ISO is a major adrenergic receptor agonist, widely used to induce heart failure models. In addition to inducing heart failure, ISO can also enhance myocardial contractility, induce cardiomyocyte death, and cause myocardial fibrosis. In addition to these effects, experiments have found that ISO can also cause myocardial mitochondrial oxidative stress. Considering the above reasons, ISO injection was selected for modeling. The results of this experiment showed that: from the analysis of echocardiogram results, the LVEDD and LVESD of the mice in the model group increased, while the LVEF and LVFS decreased; from the analysis of biomarkers, the serum BNP level in the model group increased; from the analysis of pathological results, the arrangement of cardiomyocytes in the model group was disordered, a large amount of blue collagen fibers were deposited, and myocardial fibrosis was obvious, accompanied by obvious inflammatory infiltration; from the perspective of proteins and genes, the relative expression levels of the proteins and mRNAs of the myocardial fibrosis-related factors Col-I, Col-III, and α-SMA in the myocardial tissues of the mice in the model group increased, indicating that the injection of ISO led to myocardial fibrosis in mice. At the same time, p-YAP and p-LATS1 in the model group were down-regulated, indicating that the activation of the Hippo-YAP pathway was inhibited. Compared with the model group, the protein expressions of Col-I, Col-III, and α-SMA in the traditional Chinese medicine composition capsule group were down-regulated, while p-YAP and p-LATS1 were up-regulated, indicating that the traditional Chinese medicine composition capsule activated the Hippo-YAP pathway, resulting in the degradation of YAP in the cytoplasm, and the decrease in YAP expression reduced myocardial fibrosis in mice.

[0098] The Hippo / YAP pathway is an important signal transduction system that affects cell division, proliferation, and death. The Hippo / YAP pathway is mainly used to regulate cell proliferation and apoptosis to control organ size and tissue homeostasis during animal development and regeneration. This pathway functions through the expression of multiple proteins, including Hippo kinases, mammalian sterile 20-like kinases 1 / 2 (MST1 / 2), large tumor suppressor 1 / 2 (LATS1 / 2), MOB kinase activator 1 (MOB1), Salvador family WW domain-containing protein 1 (SAV1), and YAP / TAZ. In this pathway, MST1 / 2 located upstream can bind to SAV1 to form an active enzyme, which can activate and phosphorylate LATS1 / 2. At the same time, MST1 / 2 can phosphorylate MOB1. Phosphorylated LATS1 / 2 forms a complex with MOB1, further phosphorylating downstream factors YAP / TAZ. YAP / TAZ are transcriptional coactivators that can regulate the expression of multiple genes and are the downstream effector proteins of the Hippo / YAP pathway. When YAP / TAZ are in an unphosphorylated state, they enter the nucleus and bind to TEAD family transcription factors, promoting the transcription of YAP genes. The activation of the Hippo pathway promotes the phosphorylation of YAP / TAZ, ultimately leading to the degradation of YAP / TAZ in the cytoplasm and the inhibition of YAP expression. During the process of myocardial fibrosis, the activation of the Hippo pathway is inhibited, ultimately leading to an increase in YAP expression. YAP will participate in controlling the activation of cardiac fibroblasts and fibroinflammatory responses by acting on the downstream of the Wnt and TGFβ1 signaling pathways.

[0099] The traditional Chinese medicine composition capsule is composed of red ginseng, salvia miltiorrhiza, and cornel fruit from Example 1. In this traditional Chinese medicine composition, red ginseng can tonify the heart yang qi, cornel fruit can replenish qi and secure the exterior, and salvia miltiorrhiza can promote blood circulation to remove blood stasis and clear the heart and relieve vexation. Generally speaking, the traditional Chinese medicine composition capsule can increase qi and blood, nourish yin and yang, promote blood circulation, and treat qi deficiency and blood stasis in heart failure. Its active ingredients include ginsenosides, red ginseng polysaccharides, luteolin, tanshinones, iridoids and their glycosides, etc. Ginsenosides can inhibit myocardial fibrosis by regulating the miR-489 / myd88 / NF-κB pathway and can also inhibit cardiac hypertrophy and heart failure. Luteolin can target the TLR4 / MyD88 / NF-κB signaling pathway to alleviate heart failure in rats. Tanshinones can also prevent myocardial infarction and heart failure by intervening in targets including NF-κB and AMPK.

[0100] In summary, this experiment induced and constructed a CHF mouse model by ISO and explored the improvement mechanism of the traditional Chinese medicine composition capsule on myocardial fibrosis in CHF mice. It was confirmed that the traditional Chinese medicine composition capsule can improve the heart function of CHF mice, reduce the degree of myocardial fibrosis, and decrease the expression of fibrosis-related proteins and YAP in myocardial tissues.

[0101] Clinical trial:

[0102] This protocol included 148 CHF patients with the syndrome of vital energy deficiency. They were divided into an experimental group and a control group at a ratio of 1:1, with 74 subjects in each group. The experimental group was treated with conventional Western medicine plus a traditional Chinese medicine composition capsule (i.e., the capsule prepared from the traditional Chinese medicine composition of Example 1), and the control group was treated with conventional Western medicine plus a placebo. The course of treatment was 28 days for both groups. The clinical efficacy, scores of traditional Chinese medicine syndrome elements, B-type natriuretic peptide (BNP), echocardiogram indexes, 6-minute walk distance (6MWD), Minnesota Living with Heart Failure Questionnaire (MLHFQ) scores, and safety evaluation indexes of the two groups were observed and compared.

[0103] Results: After treatment, the total clinical effective rate of the experimental group was 90.5% (67 / 74), which was higher than that of the control group (87.8%, 65 / 74), but the difference was not statistically significant (P>0.05). After treatment, the scores of traditional Chinese medicine syndrome elements in both groups decreased significantly compared with those before treatment, and the scores of the experimental group were lower than those of the control group, with a statistically significant difference (P<0.05). After treatment, the BNP levels in both groups decreased compared with those before treatment, and the experimental group had a lower level than the control group, with a statistically significant difference (P<0.05). After treatment, the left ventricular ejection fraction (LVEF) of the experimental group increased compared with that before treatment, with a statistically significant difference (P<0.05). After treatment, the left ventricular end-systolic diameter (LVESD) of the experimental group was lower than that of the control group, with a statistically significant difference (P<0.05). After treatment, the 6MWD of the experimental group was higher than that of the control group, with a statistically significant difference (P<0.05). After treatment, the MLHFQ score of the experimental group was lower than that of the control group, with a statistically significant difference (P<0.05). There was no statistically significant difference in the safety indexes between the two groups after treatment (P>0.05). No drug-related adverse reactions were found in both groups during the treatment process.

[0104] Comparison of clinical efficacy between the two groups:

[0105] After 28 days of treatment, among the subjects in the experimental group, 30 cases were markedly effective, 37 cases were effective, and 7 cases were ineffective, with a total effective rate of 90.5%; among the subjects in the control group, 16 cases were markedly effective, 49 cases were effective, 9 cases were ineffective, and the total effective rate was 87.8%. The total effective rate of the experimental group was higher than that of the control group. The difference was not statistically significant (P>0.05). The results are shown in Table 8.

[0106] Table 8 Comparison of clinical efficacy between the two groups [case(%)]

[0107]

[0108] Comparison of scores of traditional Chinese medicine syndrome elements between the two groups:

[0109] After treatment, the scores of the main symptoms, secondary symptoms, and total scores of the TCM syndrome elements in both groups decreased compared with those before treatment, and the scores in the experimental group were lower than those in the control group, with statistically significant differences (P < 0.05). The results are shown in Tables 9 and 10.

[0110] Table 9 Comparison of scores of TCM syndrome elements between the two groups before and after treatment - main symptoms (x±s, points)

[0111]

[0112] Note: Compared with before treatment in the same group, a P < 0.05; compared with after treatment in the control group, b P < 0.05.

[0113] Table 10 Comparison of scores of TCM syndrome elements between the two groups before and after treatment - secondary symptoms (x±s, points)

[0114]

[0115] Note: Compared with before treatment in the same group, a P < 0.05; compared with after treatment in the control group, b P < 0.05.

[0116] Comparison of brain natriuretic peptide (BNP) between the two groups:

[0117] Before treatment, there was no statistically significant difference in BNP levels between the two groups (P > 0.05). After treatment, the BNP levels in both groups decreased compared with those before treatment, and the levels in the experimental group were lower than those in the control group, with statistically significant differences (P < 0.05). The results are shown in Table 11.

[0118] Table 11 Comparison of BNP between the two groups before and after treatment (x±s, pg / mL)

[0119]

[0120] Comparison of cardiac echocardiogram indexes between the two groups:

[0121] After treatment, the left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) in both groups decreased compared with those before treatment (P < 0.05). After treatment, the left ventricular ejection fraction (LVEF) in the experimental group increased compared with that before treatment (P < 0.05). For the left ventricular end-systolic diameter (LVESD), before treatment, there was no statistically significant difference between the two groups (P > 0.05); after treatment, the experimental group was lower than the control group, with statistically significant differences (P < 0.05). The results are shown in Table 12.

[0122] Table 12 Comparison of cardiac echocardiogram indexes between the two groups before and after treatment (x±s)

[0123]

[0124] Note: LVEF: left ventricular ejection fraction; LVEDD: left ventricular end-diastolic diameter; LVESD: left ventricular end-systolic diameter. Compared with before treatment in this group, a P < 0.05; compared with after treatment in the control group, b P < 0.05.

[0125] Comparison of the 6-minute walking distance (6MWD) between the two groups:

[0126] There was no significant difference in 6MWD between the two groups before treatment (P > 0.05). After treatment, 6MWD in the experimental group was higher than that in the control group, with a statistically significant difference (P < 0.05). The results are shown in Table 13.

[0127] Table 13 Comparison of 6MWD between the two groups before and after treatment (x±s, m)

[0128]

[0129] Comparison of the Minnesota Living with Heart Failure Questionnaire (MLHFQ) scores between the two groups:

[0130] There was no significant difference in MLHFQ scores between the two groups before treatment (P > 0.05). After treatment, the MLHFQ score in the experimental group was lower than that in the control group, with a statistically significant difference (P < 0.05). The results are shown in Table 14.

[0131] Table 14 Comparison of MLHFQ scores between the two groups before and after treatment (x±s, points)

[0132]

[0133] Comparison of safety indicators:

[0134] Comparison of some safety indicators such as blood routine, liver and kidney function, and coagulation between the two groups before and after treatment showed no significant difference (P > 0.05), and no serious adverse reactions were found during the treatment period.

[0135] The traditional Chinese medicine composition capsules have significant therapeutic effects on patients with chronic heart failure of the type of vital energy deficiency. They can effectively relieve their clinical symptoms, improve cardiac function, enhance exercise tolerance, improve quality of life, and have high safety.

[0136] Typical cases:

[0137] Xu, male, 22 years old, with heart fatigue for more than 1 year, was hospitalized in the Department of Cardiology of our hospital. The echocardiogram during hospitalization showed: LA: 45, AO: 32, LV: 61, EF: 31%, BNP: 1250 pg / L, cardiac function class III, and obvious heart fatigue when walking on flat ground. Subsequently, the fine powder of the traditional Chinese medicine of the present invention was combined with diuretics and potassium supplementation. After 5 months of outpatient reexamination, the echocardiogram showed: LA: 32, AO: 32, LV: 47, EF: 50%. BNP: 69 pg / , cardiac function class I, and general activities and labor were not restricted.

[0138] Liu, male, 39 years old, with heart fatigue for more than 2 years, was hospitalized in the Department of Cardiology of our hospital. The echocardiogram during hospitalization showed: LA: 46, AO: 34, LV: 71, EF: 28%, BNP: 2600 pg / L, cardiac function class III, and obvious heart fatigue when walking on flat ground. Subsequently, the fine powder of the traditional Chinese medicine of the present invention was combined with diuretics and potassium supplementation. After 6 months of outpatient reexamination, the echocardiogram showed: LA: 31x43x35, AO: 34, LV: 55, EF: 48%. BNP: 109 pg / , cardiac function class I, and general activities and labor were not restricted.

[0139] The above are only the embodiments of the present invention, and common knowledge such as specific structures and / or characteristics known in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can still be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to interpret the content of the claims.

Claims

1. A traditional Chinese medicine composition for treating heart failure, characterized in that: Raw materials including the following weight components: 160 parts of red ginseng, 160 parts of cornel and 80 parts of salvia miltiorrhiza.

2. Application of a traditional Chinese medicine composition for treating heart failure in regulating the Hippo-YAP pathway.

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