Application of qi-regulating and blood-activating preparation in preparation of medicine for preventing or treating heart failure

Abstract

The invention relates to application of a qi-regulating and blood-activating preparation in preparation of a medicine for preventing or treating heart failure, and belongs to the technical field of medicine application. The qi-regulating and blood-activating preparation is prepared from fructus cinnamoii, blumea balsamifera, ligusticum wallichii and allium macrostemon, and the preparation can be a dripping pill, a capsule, a tablet, a granule or a solution. Experiments show that the qi-regulating and blood-activating preparation can effectively treat heart failure, especially heart failure after myocardial infarction, and can reduce the infarction area of the heart, improve inflammatory lesion of the heart and improve myocardial fibrosis; the proliferation of myocardial cells induced by blood oxygen deprivation can be promoted, and the apoptosis of the myocardial cells can be improved. The use is convenient;

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, and in particular to the application of qi-regulating and blood-activating preparations in the preparation of drugs for the prevention or treatment of heart failure. Background Technology

[0002] Heart failure (HF) is a syndrome characterized primarily by circulatory dysfunction caused by impaired cardiac output due to dysfunction of myocardial diastolic and / or systolic function, resulting in insufficient cardiac output to meet tissue metabolic needs, despite adequate venous return. Clinically, it is characterized by decreased cardiac output, reduced tissue blood perfusion, and venous congestion in the pulmonary and / or systemic circulation; hence, it is also known as congestive heart failure or heart insufficiency.

[0003] Heart failure (HF) is the end-stage of various heart diseases, affecting as many as 56 million people worldwide. It is a leading cause of mortality and morbidity globally, and has become a global public health problem. Myocardial infarction is one of the most common and important causes of heart failure worldwide. Although clinically used methods such as thrombolysis, percutaneous coronary intervention (PCI), and arterial bypass surgery can rapidly restore coronary blood flow, significantly reduce infarct size, and improve patient survival rates, they are insufficient to prevent cardiomyocyte apoptosis and further deterioration of cardiac function, ultimately leading to heart failure. Post-MI HF has a significantly higher mortality rate than simple myocardial infarction, with an incidence of approximately 14.3%-19.3% in acute myocardial infarction patients after PCI. In recent years, some new drugs have emerged in the treatment of heart failure, such as angiotensin receptor-neprilysin inhibitors (ARNI), guanylate cyclase stimulants, and sodium-glucose cotransporter 2 (SGLT2) inhibitors. However, most of these drugs are used in the middle and late stages of heart failure and have varying degrees of side effects. There is an urgent need for safer and more effective treatments and strategies to address the risk of heart failure after myocardial infarction.

[0004] Qi-regulating and blood-activating pills are a pure traditional Chinese medicine preparation made from Litsea cubeba, Allium macrostemon, Ligusticum chuanxiong, and Artemisia argyi. They possess aromatic and warming properties, regulate qi, and relieve pain. Clinically, they are used to prevent and treat chest pain and angina pectoris (coronary heart disease) caused by insufficient heart yang and blood stasis, with symptoms such as chest tightness, chest pain, palpitations, shortness of breath, and cold limbs. Extensive clinical use has shown that qi-regulating and blood-activating preparations are effective in treating angina pectoris. However, there are no reports on their effects in treating heart failure. Therefore, research on the prevention or treatment of heart failure and other diseases using qi-regulating and blood-activating preparations is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide the application of qi-regulating and blood-activating preparations in the preparation of drugs for the prevention or treatment of heart failure.

[0006] The heart failure includes post-myocardial infarction heart failure, and the qi-regulating and blood-activating preparation can reduce the infarct area of ​​the heart, improve inflammatory lesions of the heart, and improve myocardial fibrosis.

[0007] Furthermore, qi-regulating and blood-activating preparations can prevent or treat myocardial cell apoptosis.

[0008] Furthermore, qi-regulating and blood-activating preparations can promote the proliferation of cardiomyocytes induced by blood oxygen deprivation and improve their apoptosis.

[0009] The qi-regulating and blood-activating preparation of the present invention is made from the following raw materials in the following weight proportions: 450 parts of Litsea cubeba, 9 parts of Artemisia argyi, 360 parts of Ligusticum chuanxiong, and 36 parts of Allium macrostemon.

[0010] The qi-regulating and blood-activating preparation is prepared into the required dosage form by adding one or more pharmaceutically acceptable diluents, excipients, disintegrants, humectants, binders, flavoring agents, and antibacterial agents as needed, and following conventional methods in the pharmaceutical field.

[0011] The dosage form of the qi-regulating and blood-activating preparation is drop pills, capsules, tablets, granules, or solutions; preferably drop pills.

[0012] The qi-regulating and blood-activating preparation of this invention is prepared according to existing patent or non-patent literature technology and national drug standards. Its preparation method is prior art and can be obtained from the literature disclosed in existing patents, or prepared according to the technology and drug standards also disclosed therein. For example, it can be prepared according to the methods disclosed in YBZ00782012, CN1515303A, and CN114073740A.

[0013] The dosage and administration of the Qi-regulating and blood-activating preparations described in this invention are determined according to the patient's condition. For example, the commercially available Qi-regulating and blood-activating dripping pills have the following specifications: each pill weighs 25mg, and the dosage and administration are: 10 pills orally three times a day.

[0014] Traditional Chinese medicine believes that the core pathogenesis of heart failure is deficiency of the root and excess of the branch. The main treatment principle of traditional Chinese medicine is to warm the yang, replenish qi and consolidate the root, and promote blood circulation and remove blood stasis.

[0015] Litsea cubeba is the dried fruit of a plant in the genus Cinnamomum of the Lauraceae family. It is pungent and warm in nature, possessing properties of dispelling cold and dampness, promoting qi circulation, and relieving pain. Modern pharmacological studies have demonstrated its ability to relax smooth muscle, increase coronary blood flow, reduce myocardial oxygen consumption, improve hemodynamics, resist experimental arrhythmias, and exhibit antibacterial, antiviral, anti-inflammatory, anti-swelling, and analgesic effects. It also provides good protection against acute experimental myocardial ischemia and infarction in cats.

[0016] Ligusticum chuanxiong is the rhizome of Ligusticum chuanxiong, a plant belonging to the genus Ligustrum of the family Apiaceae. It is warm in nature, pungent and slightly bitter in taste, and is a commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis. It contains volatile oils, alkaloids, phenolic substances, organic acids, phthalyl lactones, and other components; it has the effects of promoting blood circulation, regulating qi, dispelling wind, and relieving pain. Clinical pharmacological studies have shown that Ligusticum chuanxiong has the effects of relaxing smooth muscle, increasing coronary blood flow, reducing myocardial oxygen consumption, improving myocardial metabolism, dilating peripheral blood vessels, inhibiting platelet aggregation, preventing thrombosis, increasing blood flow, improving microcirculation, and providing sedation and analgesia.

[0017] Allium macrostemon B. or Allium chinensis G. Don, belonging to the genus Allium of the Liliaceae family, is a dried bulbous plant. It is pungent and bitter in nature, and has the effects of warming the middle jiao and promoting yang, regulating qi and relieving chest tightness, and dispersing stagnation. Clinically, Allium macrostemon is often used for chest pain, heart pain radiating to the back, etc. Current clinical research shows that Allium macrostemon has the effects of inhibiting platelet aggregation, lowering lipids and preventing atherosclerosis, antibacterial and antioxidant effects, anticancer effects, antispasmodic and antiasthmatic effects, analgesic effects, and hypoxia tolerance effects.

[0018] Artemisia argyi is a processed product of the resin of the Dipterocarpus santalinus plant (family Dipterocarpaceae), or a crystal extracted from the leaves of the Asteraceae plant Artemisia argyi, or a refined product synthesized chemically using turpentine, camphor, and other raw materials. Artemisia argyi is pungent, bitter, and slightly cold in nature, and enters the heart, spleen, and lung meridians. Its main component is levorotatory borneol, and it has the functions of opening the orifices, refreshing the mind, clearing heat, and relieving pain. Modern pharmacological studies have proven that artemisia argyi has anti-myocardial ischemia effects and can significantly increase coronary blood flow; in addition, artemisia argyi can increase the permeability of the blood-brain barrier and enhance the ability of drugs to cross the barrier.

[0019] The qi-regulating and blood-activating preparation made from Litsea cubeba, Allium macrostemon, Ligusticum chuanxiong, and Artemisia argyi has the effects of warming yang, relieving chest tightness, regulating qi and activating blood. It is used for angina pectoris in coronary heart disease and also conforms to the treatment principles of traditional Chinese medicine for heart failure.

[0020] Studies have shown that the Qi-regulating and blood-activating dripping pills of this invention can significantly reduce the infarct area in rat hearts; significantly improve inflammatory lesions in the heart of rats with heart failure after myocardial infarction; significantly improve myocardial fibrosis and ventricular remodeling in rats with heart failure after myocardial infarction; and significantly reduce apoptosis of cardiomyocytes in the myocardial tissue of rats with heart failure after myocardial infarction. Simultaneously, the Qi-regulating and blood-activating dripping pills can effectively restore cell morphology under oxygen deprivation conditions, significantly increase cardiomyocyte proliferation, reduce G1 phase arrest of cardiomyocytes caused by ischemia and hypoxia, and lower the apoptosis rate of cardiomyocytes, thereby achieving the purpose of effectively preventing or treating heart failure. Compared with existing products for treating heart failure, the Qi-regulating and blood-activating preparation of this invention has advantages such as better efficacy, convenient use, and no toxic side effects, and is worthy of widespread application. Attached Figure Description

[0021] Figure 1 Effects of Qi-regulating and Blood-activating Dripping Pills on Cardiac Function, Ejection Fraction, Fractional Shortening of Heart, and Ventricular Wall Thickness in Rats with Heart Failure After Myocardial Infarction (A, B) Figure 2 Effects of Qi-regulating and Blood-activating Dripping Pills on the Infarct Area in Rats with Heart Failure After Myocardial Infarction Figure 3 Effects of Qi-regulating and Blood-activating Dripping Pills on Cardiac Pathological Changes in Rats with Heart Failure After Myocardial Infarction Figure 4 Effects of Qi-regulating and Blood-activating Dripping Pills on Myocardial Fibrosis in Rats with Heart Failure After Myocardial Infarction Figure 5 Effects of Qi-regulating and Blood-activating Dripping Pills on Apoptosis of Cardiac Tissue Cells in Rats with Heart Failure After Myocardial Infarction Figure 6 Effects of Qi-regulating and Blood-activating Dripping Pills on the Survival Rate of H9C2 Cells Induced by Blood Oxygen Deprivation Figure 7 Effects of Qi-regulating and Blood-activating Dripping Pills on the Morphology of H9C2 Cells Induced by Blood Oxygen Deprivation Figure 8 Effects of Qi-regulating and Blood-activating Dripping Pills on Oxygen Deprivation-Induced Proliferation of H9C2 Cells Figure 9 Effects of Qi-regulating and Blood-activating Dripping Pills on Oxygen Deprivation-Induced Cell Cycle in H9C2 Cells Figure 10 Effects of Qi-regulating and Blood-activating Dripping Pills on the Apoptosis Rate of H9C2 Cells Induced by Blood Oxygen Deprivation Figure 11 Effects of Qi-regulating and Blood-activating Dripping Pills on Apoptosis-related Proteins in H9C2 Cells Induced by Blood Oxygen Deprivation Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the following specific embodiments further illustrate the application of qi-regulating and blood-activating preparations in the preparation of drugs for the prevention or treatment of heart failure.

[0023] Example 1: In vivo animal experiment study on the effects of Qi-regulating and blood-activating dripping pills on heart failure after myocardial infarction. Eight-week-old male SD rats were selected, and a myocardial infarction model was established by ligating the left anterior descending coronary artery (LAD). Three days later, cardiac function and ejection fraction were evaluated using small animal ultrasound to determine the success of the model. Rats with an ejection fraction <45% were considered to have successfully developed a heart failure model after myocardial infarction. The model rats were then randomly divided into a model group (M), low-, medium-, and high-dose groups of Liqi Huoxue Diwan (L / Z / G, 43.75 / 87.5 / 175 mg / kg), and a Sham group. The drug groups were administered the corresponding drugs by gavage for two consecutive weeks. After the experiment, changes in cardiac function and ejection fraction were detected using small animal ultrasound. TTC staining was used to detect myocardial infarction, HE staining and Masson staining were used to observe cardiac tissue lesions and myocardial fibrosis, and TUNEL staining was used to observe cardiac tissue cell apoptosis.

[0024] (1) Echocardiographic examination Two weeks after modeling, echocardiography was performed before sacrifice. The hair on the front of the rats' chest was removed beforehand, and they were anesthetized with 1.25% aphthine, fixed on an ultrasound board, and subjected to echocardiography. Left ventricular short-axis rate (FS), left ventricular ejection fraction (EF), left ventricular posterior wall end-diastolic thickness (LVPWd), left ventricular end-systolic diameter (LVIDs), stroke volume (SV), and end-systolic interventricular septal thickness (IVSs) were measured over three consecutive cardiac cycles. Data were processed and statistically analyzed.

[0025] Experimental results showed that the Qi-regulating and Blood-activating Dripping Pills significantly improved cardiac function in rats, including increasing ejection fraction, stroke volume, and the ratio of diastolic to systolic thickness of the left ventricular posterior wall. (See [link to relevant documentation]) Figure 1 .

[0026] (2) TTC staining After removing the heart through thoracotomy, rinse with pre-cooled PBS until no blood adheres, dry with sterile gauze, remove surface tissue, and flash-freeze at -20°C for 30 min for easy sectioning. Cut the heart into 2-3 mm thick sections along the transverse direction from the ligation suture. Immerse the sections in 2% TTC stain for 30 min in the dark, fix with 4% paraformaldehyde solution for 24 h, blot dry the surface fluid, and photograph for observation.

[0027] The results showed that the Qi-regulating and Blood-activating Dripping Pills significantly reduced the infarct area in the heart of rats. (See...) Figure 2 .

[0028] (3) H&E staining After removing the heart via thoracotomy, rinse with pre-cooled PBS until no blood adheres, dry with sterile gauze, remove surface tissue, and fix the heart in 4% paraformaldehyde solution. Dewax paraffin sections to water (sequentially immersing sections in environmentally friendly dewaxing solution I for 15 min, environmentally friendly dewaxing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinsing with tap water). For frozen sections, thaw and fix: remove frozen sections from -20°C and bring to room temperature, fix with acetone for 1 min, then rinse with water. Treat sections with high-resolution constant staining pretreatment solution for 1 min. Stain sections with hematoxylin solution for 3 min, rinse with tap water, differentiate with differentiation solution for 5 s, rinse with tap water, re-blue with blue solution for 5 s, and rinse with running water. Dehydrate sections with 95% ethanol for 1 min, then stain with eosin solution for 15 s. The sections were sequentially immersed in anhydrous ethanol I for 1 min, then anhydrous ethanol II for 1 min, then anhydrous ethanol III for 1 min, then n-butanol I for 1 min, then n-butanol II for 1 min, then xylene I for 1 min, and finally xylene II for 1 min. After clearing, the sections were mounted with neutral resin. The sections were then examined under a microscope, and images were acquired and analyzed.

[0029] The results showed that the Qi-regulating and Blood-activating Dripping Pills significantly improved the inflammatory lesions in the heart of rats with heart failure after myocardial infarction. (See...) Figure 3 .

[0030] (4) Masson staining After removing the heart via thoracotomy, rinse with pre-cooled PBS until no blood adheres, dry with sterile gauze, remove surface tissue, and fix the heart in 4% paraformaldehyde solution. Dewax paraffin sections, freeze at -20°C, and allow to return to room temperature. Fix with tissue fixative for 15 min, then rinse with running water. Immerse sections in Masson A solution in a 65°C oven for 30 min, then rinse with running tap water. Simultaneously, preheat Masson D and Masson F solutions in a 55°C water bath. Immerse sections in a mixture of equal parts Masson B and Masson C solutions for 1 min, then rinse with tap water. Differentiate in differentiation solution for a few seconds, then rinse with tap water. Immerse sections in Masson D solution for 6 min, then rinse with tap water. Immerse in Masson E solution for 1 min, do not rinse, drain slightly, and then directly stain in Masson F solution for 30 s. The sections were rinsed and differentiated with 1% acetic acid, then dehydrated in anhydrous ethanol. The sections were then placed in anhydrous ethanol for 5 minutes, followed by xylene for 5 minutes for clearing, and finally mounted with neutral resin. Microscopic examination and image acquisition and analysis were performed.

[0031] The results showed that the Qi-regulating and Blood-activating Dripping Pills significantly improved myocardial fibrosis and ventricular remodeling in rats with heart failure after myocardial infarction. (See...) Figure 4 (5) Tnuel staining After the heart was removed via thoracotomy, it was rinsed with pre-cooled PBS until no blood adhered, dried with sterile gauze, and the surface tissue of the heart was removed. The heart was then fixed by immersion in 4% paraformaldehyde solution. Paraffin sections were dewaxed and hydrated, and antigen retrieval was performed at 37°C for 20 min using proteinase K working solution. Subsequently, TUNEL reaction solution was prepared according to the kit instructions (Servicebio, China), added to the tissue area, and incubated at 37°C in the dark for 1 h to label the broken DNA ends of apoptotic cells. After incubation, the cell nuclei were counterstained with DAPI staining solution. Finally, the sections were mounted with anti-fluorescence quenching mounting medium, observed under a fluorescence microscope, and images were acquired.

[0032] The results showed that the Qi-regulating and Blood-activating Dripping Pills significantly reduced cardiomyocyte apoptosis in the myocardial tissue of rats with heart failure after myocardial infarction. (See...) Figure 5 .

[0033] Example 2: In vitro cell experiment study on the effect of Qi-regulating and blood-activating dripping pills on oxygen deprivation-induced cardiomyocyte proliferation. H9C2 cells that have grown to 80% were removed from the incubator, the supernatant was collected, washed with PBS, and then replaced with serum-free culture medium. After synchronization for 6 hours, the cells were placed in a three-gas incubator with an O2 concentration of ≤1% for 12 hours of oxygen deprivation to establish an oxygen deprivation (HI) model.

[0034] The experiment was divided into a normal group (Ctrl.) and a group treated with Liqi Huoxue Dripping Pills (L / Z / G, 1 / 5 / 10 μg / mL). H9C2 cells were cultured under oxygen deprivation when their density reached 80-90%, and then incubated with different concentrations of Liqi Huoxue Dripping Pills for 12 h. The absorbance was measured at 450 nm using the MTT assay to calculate cell viability. Cell morphology was observed using Giemsa staining, cell proliferation was observed using the scratch assay, cell cycle arrest was detected using Annexin V-PI staining, cardiomyocyte apoptosis was detected using Annexin V-FITC / PI double staining, and the expression of apoptosis-related proteins was detected using Western blot.

[0035] (1) Cell culture, cryopreservation and thawing Prepare complete culture medium by mixing 89% high-glucose DMEM, 10% fetal bovine serum, and 1% antibiotics. Seed 3 ml of the mixture into a T25 culture flask and incubate at 37°C with 5% CO2. Observe cell status and density under an inverted microscope every 12 hours, replacing the complete culture medium as needed. Once the cell density reaches 80% or higher, other procedures can be performed.

[0036] Take a T25 culture flask with a cell density of 80% or higher and place it in a UV-sterilized clean bench. Discard the original culture medium in the flask, add 2 mL of PBS to wash, and discard the liquid. Add 1 mL of 0.25% trypsin for digestion. When the cells detach from the bottom of the flask, add 2 mL of complete culture medium to stop the digestion. Collect the liquid into a 5 mL round-bottom centrifuge tube, centrifuge at 1000 rpm for 5 min, remove and place in a clean bench, aspirate the supernatant, and keep the cell pellet. Add 1 mL of complete culture medium to the centrifuge tube and gently mix. Take 0.5 mL and add it to a T25 culture flask containing 3 mL of complete culture medium. Tightly cap the flask, place it flat, and gently shake to distribute the cells evenly. Incubate at 37°C in a 5% CO2 incubator. Observe under a microscope and change the medium as needed.

[0037] Take a T25 culture flask with a cell density of 80% or higher and place it in a UV-sterilized clean bench. Discard the original culture medium in the flask, add 2 mL of PBS to wash, and discard the liquid. Add 1 mL of 0.25% trypsin for digestion. When the cells detach from the bottom of the flask, add 2 mL of complete culture medium to stop the digestion. Collect the liquid into a 5 mL round-bottom centrifuge tube and centrifuge at 1000 rpm for 5 min. After discarding the supernatant, add 1 mL of cell cryopreservation solution, mix well, and transfer to a cryovial. Label the cell line name, date, cell passage number, and operator's name. Incubate overnight at -80°C and store in liquid nitrogen.

[0038] Remove the cryovials from the cells and thaw them in a 37°C water bath until they are approximately 70% thawed. Stop the water bath and transfer the cells to a 5 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. Discard the supernatant; resuspend the cells in 1 mL of complete culture medium and transfer the resuspended cells to a T25 culture flask pre-filled with 3 mL of complete culture medium and placed in an incubator at a constant temperature. Gently shake the flask, observe under a microscope, and then incubate at 37°C in a 5% CO2 incubator.

[0039] (2) Establishment of cell model H9C2 cells in their growth phase were selected and seeded into 96-well plates (100 μL per well) using serum-free and glucose-free medium. The plates were incubated at 37°C with 5% CO2 until cell adhesion was achieved. The plates were then placed in a tri-gas incubator with 1% O2 for further incubation. Odulation was assessed at 4, 8, 12, 24, 36, and 48 h of hypoxia, and the OD values ​​of each well were recorded using the MTT assay. 490 The H9c2 cell viability was calculated, and the optimal hypoxia modeling concentration was determined when the cell viability dropped to 80%. The optimal hypoxia modeling time was then used to screen for hypoxia model initiation. The selected hypoxia modeling time was then recorded by measuring the OD of each well using the MTT assay. 490 H9c2 cell viability was calculated, and low, medium, and high dose concentrations of the Qi-regulating and Blood-activating Dripping Pill were screened. For ischemia and hypoxia, the determined modeling time and drug concentration were used, and the OD of each well was recorded by MTT assay. 490 The survival rate of H9C2 cells was calculated to evaluate the efficacy of Liqi Huoxue Diwan on ischemia-hypoxia-induced H9c2 cell damage.

[0040] (3) MTT method H9c2 cells in logarithmic growth phase and in good condition were digested with 0.25% trypsin and then prepared into a single-cell suspension using complete culture medium. Cells were counted using a hemocytometer, and the cell suspension was seeded at 100 µL per well in a 96-well plate, resulting in approximately 5 × 10³ cells / well. The culture plates were incubated at 37°C with 5% CO2 for 24 h to allow for full cell adhesion. Grouping and drug treatment were performed 4 h before the scheduled treatment time in a clean bench. 20 µL of MTT solution (prepared with PBS to a 5 mg / mL concentration, filtered through a 0.22 µm filter for sterilization) was added to each well. The culture plate was gently shaken to mix, and then returned to the incubator for another 4 h in the dark. After incubation, the supernatant in each well was carefully aspirated. 150 µL of LDMSO was added to each well, and the culture plate was placed on a horizontal shaker and shaken at low speed for 10 min to fully dissolve the blue-purple formazan crystals at the bottom of the plate. The absorbance of each well was measured at a wavelength of 490 nm using an ELISA reader, and the data were processed and statistically analyzed.

[0041] The results showed that, at an effective concentration of 1-10 µg / mL, the Qi-regulating and Blood-activating Dripping Pills significantly increased the survival rate of cardiomyocytes. (See...) Figure 6 .

[0042] (4) Giemsa staining Cells in the logarithmic growth phase were seeded into six-well plates, treated with the drug, and then gently washed with PBS. The PBS was discarded, and the cells were fixed with methanol for 15 min, followed by air drying at room temperature. Freshly prepared Giemsa working solution (Giemsa stock solution: pH 6.8 phosphate buffer at a volume ratio of 1:9, freshly prepared and filtered) was added to the six-well plates, and the cells were stained at room temperature for 20 min. After washing and soaking with PBS, cell morphology was observed under an optical microscope.

[0043] The results showed that the Qi-regulating and blood-activating dripping pills could effectively restore cell morphology under conditions of blood oxygen deprivation. (See...) Figure 7 .

[0044] (5) Cell scratch test Cells in logarithmic growth phase were seeded into six-well plates and treated with medication. Once the cells reached nearly 100% confluence into a monolayer, a sterile 200 μL pipette tip was used to make straight incisions along a ruler on the monolayer of cells, perpendicular to the bottom of the plate. The plates were gently washed three times with PBS to remove detached cells. The medium was replaced with serum-free medium, and three fixed locations along the edge of the incisions were marked and photographed under an inverted microscope, recorded as 0 h. The plates were then returned to a 37°C, 5% CO2 incubator for further culture. Photographs were taken again at the same marked locations at 4, 8, 12, and 24 h post-culture. The width of the incisions was measured using ImageJ software, cell migration was calculated, and statistical analysis was performed.

[0045] The results showed that the Qi-regulating and blood-activating dripping pills significantly increased cardiomyocyte proliferation under oxygen deprivation conditions. (See...) Figure 8 .

[0046] (6) Cell cycle detection Cells in logarithmic growth phase were seeded into six-well plates. After drug treatment, cells were collected from each group by digestion with 0.25% trypsin and washed twice with pre-chilled PBS. Cells were resuspended in 1 mL PBS, and 3 mL of pre-chilled 70% ethanol was slowly added. The mixture was vortexed and fixed overnight at 4°C. After fixation, the cells were centrifuged to remove the ethanol and washed once with PBS. PI staining solution (final concentration 50 μg / mL) was added, and the cells were incubated in the dark for 30 min. Flow cytometry was used for detection, with an excitation wavelength of 488 nm, and the fluorescence signal of the PE channel was collected. The percentage of cells in G0 / G1, S, and G2 / M phases was analyzed and statistically determined.

[0047] The results showed that, at effective concentrations, the Qi-regulating and Blood-activating Dripping Pills significantly reduced G1 phase arrest of cardiomyocytes caused by ischemia and hypoxia. (See...) Figure 9 .

[0048] (7) Apoptosis detection Cells in logarithmic growth phase were seeded into six-well plates. After drug treatment, cells from each group were collected by trypsin-free digestion and washed twice with pre-chilled PBS. Cells were resuspended in 100 μL of 1× Binding Buffer. 5 μL of Annexin V-FITC and 5 μL of PI staining solution were added sequentially, mixed well, and incubated at 37°C in the dark for 15 min. After incubation, 400 μL of 1× Binding Buffer was added to each tube, mixed well, and placed on ice. Fluorescence signals in the FITC and PE channels were collected using flow cytometry. Unstained cells served as negative controls, and cells stained with Annexin V-FITC and PI alone served as fluorescence compensation controls. The apoptosis rate in each group was analyzed and statistically analyzed.

[0049] The results showed that the Qi-regulating and Blood-activating Dripping Pills significantly improved cardiomyocyte apoptosis and reduced its apoptosis rate. (See [link to product]). Figure 10 .

[0050] (8) Western blot experiment Cells in logarithmic growth phase were seeded into six-well plates, treated with the drug, washed twice with PBS, and lysed thoroughly with RIPA lysis buffer. Total protein was extracted, and protein concentration was determined using the BCA method. The cells were then prepared into gels, subjected to electrophoresis, transferred to membranes, and blocked. Incubation was performed with Bax, Bcl-2, and Caspase 9, with β-ACTIN as an internal control. The reaction was carried out overnight at 4°C. After washing with TBST, HRP-labeled goat anti-mouse IgG or HRP-labeled goat anti-rabbit IgG was added as a secondary antibody. The reaction was carried out with slow shaking at room temperature for 2 h. After washing, ECL luminescence was developed, and the images were exposed and photographed. The grayscale values ​​of the images were analyzed using ImageJ software.

[0051] The results showed that Liqi Huoxue Dripping Pills could improve cardiomyocyte apoptosis by regulating the expression of apoptosis-related proteins. (See...) Figure 11 .

Claims

1. Application of Qi-regulating and blood-activating preparations in the preparation of drugs for the prevention or treatment of heart failure.

2. Application of Qi-regulating and blood-activating preparations in the preparation of drugs for the prevention or treatment of heart failure after myocardial infarction.

3. Application of Qi-regulating and blood-activating preparations in the preparation of drugs for the prevention or treatment of cardiomyocyte apoptosis.

4. The application according to claim 2, characterized in that, The qi-regulating and blood-activating preparations can reduce the infarct area of ​​the heart, improve inflammatory lesions of the heart, improve myocardial fibrosis, and reduce myocardial cell apoptosis.

5. The application according to claim 3, characterized in that, The qi-regulating and blood-activating preparation can promote the proliferation of cardiomyocytes induced by blood oxygen deprivation and improve their apoptosis.

6. The application according to any one of claims 1-5, characterized in that, The qi-regulating and blood-activating preparation is prepared by extracting 450 parts of Litsea cubeba, 9 parts of Artemisia argyi, 360 parts of Ligusticum chuanxiong, and 36 parts of Allium macrostemon.

7. The application according to any one of claims 1-5, characterized in that, The qi-regulating and blood-activating preparations are available in the form of pills, capsules, tablets, granules, and solutions.

8. The application according to claim 7, characterized in that, The qi-regulating and blood-activating preparation is a drop pill.

9. The application as described in any one of claims 1-5, characterized in that, The qi-regulating and blood-activating preparation is prepared into the required dosage form by adding one or more pharmaceutically acceptable diluents, excipients, disintegrants, humectants, binders, flavoring agents, and antibacterial agents as needed, and following conventional methods in the pharmaceutical field.

10. The application according to any one of claims 1-5, characterized in that, The administration route of the qi-regulating and blood-activating preparation is oral.

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