Application of musk Tongxin dropping pill in medicine for treating cardiovascular diseases and medicine containing musk Tongxin dropping pill

Abstract

The invention belongs to the technical field of medicine application and development, and particularly relates to application of a musk heart-dredging dropping pill. The musk heart-dredging dropping pill is used for treating and / or relieving cardiovascular diseases, and the cardiovascular diseases comprise inflammation and / or cell hypoxia injury caused by cardiovascular lesion and / or injury and / or chronic heart failure. The musk heart-smoothing dropping pill can also be used for preparing medicines and / or health-care foods for improving inflammation and / or cell hypoxia injury caused by cardiovascular diseases and / or injury and / or chronic heart failure. The invention provides a new application field and direction of the musk heart-dredging dropping pill, develops the application of the musk heart-dredging dropping pill, and enables the musk heart-dredging dropping pill to have the value of further processing for indirect application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application development technology, and particularly relates to the application of Musk Tongxin Dripping Pills in the treatment of cardiovascular diseases, and to drugs containing Musk Tongxin Dripping Pills. Background Technology

[0002] Musk Heart-Clearing Pills are a traditional Chinese medicine composed of seven medicinal materials, including artificial musk, total saponins from ginseng stems and leaves, toad venom, salvia miltiorrhiza, artificial bezoar, bear bile powder, and borneol. Its development stems from the inheritance and innovation of traditional Chinese medicine. The formula originates from the "Zhibao Dan" in the Song Dynasty's *Taiping Huimin Heji Jufang*, and has been modified based on modern pharmacological research. Modern pharmacological studies have found that Musk Heart-Clearing Pills have a multi-target mechanism of action, including protecting cardiomyocytes, inhibiting inflammatory responses, resisting oxidative stress, protecting vascular endothelium, improving coronary microcirculation, and anti-atherosclerosis. It has the effects of aromatic invigorating qi and clearing the meridians, promoting blood circulation, removing blood stasis, and relieving pain. It is mainly used for stable exertional angina pectoris due to qi deficiency and blood stasis in coronary heart disease.

[0003] As modern medicine gains a deeper understanding of cardiovascular diseases, the multi-target mechanism of action and comprehensive therapeutic advantages of Musk Tongxin Dripping Pills provide new ideas and methods for exploring their new applications. Summary of the Invention

[0004] To address the limited application areas of existing musk-containing heart-clearing pills, and the fact that many applications still need to be developed, this invention provides the application of musk-containing heart-clearing pills in the treatment of cardiovascular diseases, as well as corresponding drugs containing musk-containing heart-clearing pills.

[0005] The main objective of this invention is: I. Providing new application areas for Musk Heart-Clearing Drops; II. Provide medicines containing musk-containing Tongxin dripping pills with specific functions.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] Application of Musk Heart-Clearing Drops in the Treatment of Cardiovascular Diseases.

[0008] Preferably, the cardiovascular disease includes inflammation and / or cellular hypoxia-induced damage caused by cardiovascular lesions and / or injury, and / or chronic heart failure.

[0009] As a preferred option The treatment of cardiovascular disease includes inhibiting the release of endogenous nitric oxide.

[0010] As a preferred option The treatment of cardiovascular disease includes improving the survival rate of HUVECs cells.

[0011] As a preferred option The treatment of cardiovascular diseases includes improving cardiac function indicators; The cardiac function indicators include CO and / or LVEF and / or LVF.

[0012] Medicines containing musk-containing Tongxin dripping pills, The drug contains raw materials including musk-containing Tongxin pills; The drug has the function of treating cardiovascular diseases; The treatment of cardiovascular diseases includes inhibiting the release of endogenous nitric oxide and / or increasing the survival rate of HUVECs cells and / or improving cardiac function indicators; The cardiac function indicators include CO and / or LVEF and / or LVF.

[0013] As a preferred option The drug also includes one or more other pharmaceutically acceptable carriers and / or excipients.

[0014] As a preferred option The drug in question is a commonly used pharmaceutical preparation in pharmaceutics.

[0015] As a preferred option The pharmaceutical preparation is a tablet and / or capsule and / or granule and / or powder and / or liquid preparation.

[0016] As a preferred option The drug is administered orally and / or by injection.

[0017] Within the framework of this invention, network pharmacology technology is used to construct a component-target network for Shexiang Tongxin Dripping Pills. During the construction process, various authoritative database resources were extensively collected, including databases of traditional Chinese medicine components, disease-related gene databases, and protein-protein interaction databases. Utilizing professional algorithms and software tools, the potential targets corresponding to each component of Shexiang Tongxin Dripping Pills were accurately identified and integrated, completing the component-target network construction. Furthermore, based on this network, biological functional analysis was conducted using systems biology and bioinformatics methods. Through enrichment analysis, pathway mapping, and other techniques, the biological processes, cellular components, and molecular functions involved in this network were thoroughly analyzed to explore the potential mechanism of action of Shexiang Tongxin Dripping Pills.

[0018] Based on this, the inflammatory response and vascular endothelial cell damage, which are prominent in the biological function analysis of disease network regulatory genes, will be established as the basis for evaluating the efficacy of Shexiang Tongxin Dripping Pills in treating cardiovascular diseases. Inflammation plays a crucial role in the occurrence and development of cardiovascular diseases; excessive inflammatory activation exacerbates vascular wall damage and promotes the formation and development of atherosclerotic plaques. Vascular endothelial cell damage, as a key early pathological change in cardiovascular diseases, can trigger a series of vascular dysfunctions. Therefore, effective components will be screened targeting these two key aspects.

[0019] To accurately screen the main effective components of Shexiang Tongxin Dripping Pills for cardiovascular disease and fully validate them through in vivo experiments, a RAW264.7 mononuclear macrophage inflammation model and a HUVECs umbilical vein endothelial cell OGD (oxygen-glucose deprivation) model were used. During the experiment, the inflammatory response was first observed using a lipopolysaccharide (LPS)-induced RAW264.7 mononuclear macrophage inflammation model. As a major component of the cell wall of Gram-negative bacteria, LPS can specifically activate mononuclear macrophages, triggering a strong inflammatory response and simulating the in vivo inflammatory microenvironment. Based on this model, the inhibition rate of nitric oxide (NO) release was used as an indicator to screen the effective components of Shexiang Tongxin Dripping Pills with anti-inflammatory effects. Nitric oxide, as an important inflammatory mediator, plays a role in signal transduction and regulation in the inflammatory response, and its excessive release is closely related to the occurrence and development of inflammation. By accurately measuring the nitric oxide release inhibition rate, the intervention efficacy of each component of Shexiang Tongxin Dripping Pills on the inflammatory response was quantitatively evaluated. In the experiment, multiple drug concentration gradients were used to treat RAW264.7 cells, with a control group also included. The Griess reagent method was used to characterize nitrite levels, and the nitric oxide release inhibition rate was calculated by measuring the nitric oxide content in the cell culture supernatant. By comparing data from different treatment groups, the effective components with significant anti-inflammatory activity were identified.

[0020] Secondly, to address vascular endothelial cell injury, an OGD model of HUVECs (humid vein endothelial cells) was used. This model simulates an ischemic and hypoxic environment, subjecting umbilical vein endothelial cells to oxygen and glucose deprivation, inducing cell damage, and reproducing the pathological state of vascular endothelial cells during cardiovascular disease. Under this model, cell viability was used as an indicator to screen the main effective components of Shexiang Tongxin Dripping Pills against vascular endothelial hypoxia-induced injury. Cell viability, as a key parameter reflecting cell vitality and function, can directly demonstrate the protective effect of drug components on damaged vascular endothelial cells. In the experiment, HUVECs cells were placed in a specific hypoxic incubator, and the oxygen and glucose concentrations were adjusted to the preset hypoxic and hypoglycemic conditions. At the same time, different concentrations of Shexiang Tongxin Dripping Pills were added for intervention. Cell viability was detected at specific time points using detection methods (MTT method, CCK-8 method). By comparing the differences in cell viability between different component treatment groups and the normal control group and the model control group, the effective components with anti-vascular endothelial hypoxia-induced injury effects were identified.

[0021] Finally, to further verify the actual effects of the active ingredient in the in vivo environment, a mouse model of chronic heart failure was used. This model induces myocardial infarction in mice through surgical ligation of the left anterior descending coronary artery, leading to chronic heart failure, effectively simulating the pathophysiological process of human chronic heart failure. In this model experiment, monomeric components that showed excellent anti-inflammatory activity and anti-endothelial cell hypoxia injury effects were selected for validation studies. Mice were randomly divided into multiple groups, including a normal control group, a model control group, a positive drug control group, and groups treated with different monomeric components. The treatment groups were continuously administered the corresponding monomeric components to mice for a period of time according to the set dosage and administration method. During this period, echocardiography and hemodynamic monitoring were used to dynamically monitor the cardiac function indicators of mice; blood samples and cardiac tissue samples were collected simultaneously, and biochemical analysis and immunohistochemical experiments were used to detect the levels of inflammatory factors, vascular endothelial function-related indicators, and cardiomyocyte apoptosis. By comprehensively evaluating the therapeutic effects of the selected monomeric components on cardiovascular diseases at the whole organism level, the effectiveness of the active ingredient was verified from multiple dimensions.

[0022] The beneficial effects of this invention are: This invention provides new application areas and directions for Musk Tongxin Dripping Pills, expands the uses of Musk Tongxin Dripping Pills, and gives them the value of further processing for indirect applications. Attached Figure Description

[0023] Figure 1 The target network of components in Musk Tongxin Dripping Pills; Figure 2 Results of biological function analysis of the target sites of components in Musk Tongxin Drops; Figure 3The effect of administration of the main component 24 h on the survival rate of RAW264.7 cells was characterized. Figure 4 The results characterize the effect of the main components on the LPS-induced nitric oxide release rate in RAW264.7 cells. Figure 5 The effect of the main components on the survival rate of HUVECs cells; Figure 6 The effect of the main components on the survival rate of hypoxic-damaged HUVECs, compared with the model group (Model). * P<0.05; Figure 7 To investigate the effect of different concentrations of salvianolic acid C solution on the survival rate of hypoxic-induced HUVECs, compared with the model group (Model). * P<0.05; Figure 8 Representative echocardiograms of mice after administration of salvianolic acid C, n=5; Figure 9 The effect of six weeks of administration of salvianolic acid C on cardiac function in mice with chronic heart failure, n=5; Figure 10 Effect of administration of musk-containing Tongxin dripping pills for 24 hours on the survival rate of RAW264.7 cells; Figure 11 The effect of musk-infused Tongxin dripping pills on LPS-induced nitric oxide release rate in RAW264.7 cells; Figure 12 The effect of musk-containing Tongxin dripping pills on the survival rate of HUVECs cells; Figure 13 The effect of musk-infused heart-clearing pills on the survival rate of hypoxic-damaged HUVECs cells. ** P<0.01; in, Figure 8 and Figure 9 In the study: Sham: sham surgery group; Model: model group; DC: salvianolic acid C group. Detailed Implementation

[0024] The present invention will be further described clearly and in detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, all raw materials used in the embodiments of the present invention are commercially available or obtainable by those skilled in the art; unless otherwise specified, all methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0026] Example 1 Establish a target network for the components of Musk Tongxin Droplets and identify the main screening targets: A total of 307 target sites of the main components of Musk Tongxin Dripping Pills were integrated from ETCM (http: / / www.tcmip.cn / ETCM / index.php / Home / Index / index.html) and predicted by BATAM-TCM (http: / / bionet.ncpsb.org.cn / batman-tcm / ), and imported into Cytoscape V3.8.0 software for network visualization. Subsequently, Metascape was used for biological function analysis, and cell models were selected for screening of pharmacodynamic substances based on the analysis results and the previous pathway analysis results.

[0027] Based on previous research results on the chemical composition of Musk Tongxin Dripping Pills and data from our research group, we integrated the target results from ETCM (http: / / www.tcmip.cn / ETCM / index.php / Home / Index / index.html) and BATAM-TCM (http: / / bionet.ncpsb.org.cn / batman-tcm / ) with a prediction score greater than 20. This yielded 307 known target sites for the 23 components in Musk Tongxin Dripping Pills. These targets were then visualized using Cytoscape V3.8.0 software, resulting in a preliminary target network for the components of Musk Tongxin Dripping Pills. Figure 1 , Figure 1 The innermost circle represents the components, and the outer three circles represent the targets. Subsequently, biological functional analysis using Metascape showed that, for example... Figure 2 The active ingredients of Musk Tongxin Dripping Pills may exert their anti-cardiovascular disease effects by regulating inflammatory responses, oxidative stress, and cAMP. Based on the results of previous biological function analysis of disease network regulatory genes, RAW264.7 cell inflammation model and HUVECs cell hypoxia injury model were selected for screening of active ingredients.

[0028] Based on previous research results on the chemical composition of Musk Tongxin Dripping Pills and the research data of our research group, 16 monomers were selected as the main components of Musk Tongxin Dripping Pills for screening of pharmacodynamic substances, including bufotoxin ester, tanshinone IIA, salvianolic acid C, muscone, bufotoxin, cholic acid, deoxycholic acid, bufotoxin, ursodeoxycholic acid, deacetylated bufotoxin, far-infrared bufotoxin, taurocholic acid, chenodeoxycholic acid, glycocholic acid, ginsenoside Rg1, and ginsenoside Rk3.

[0029] Preparation of cell experiment solutions: Complete DMEM culture medium: Prepare complete DMEM culture medium by mixing high glucose DMEM medium, heat-inactivated fetal bovine serum, and double antibiotic solution in a ratio of 89:10:1.

[0030] Preparation of storage solutions for different monomers: Take an appropriate amount of monomer powder and prepare storage solutions with a concentration of 400mM or 200mM. Dispense the solutions for use and dilute to a final concentration of 400μM or the corresponding target concentration before use.

[0031] Preparation of Lipopolysaccharide (LPS) stock solution: Accurately weigh 10.0 mg LPS powder, add PBS to a final volume of 10 mL, and prepare an LPS stock solution with a concentration of 1 mg / mL. Aliquot the solution for use. When using, dilute with DMEM complete medium to a final concentration of 200 ng / mL.

[0032] Preparation of dexamethasone solution: Accurately weigh 2.0 mg of dexamethasone powder, add an appropriate amount of PBS, and prepare a solution with a concentration of 1.276 μM. Aliquot the solution for use.

[0033] RAW264.7 cell culture and passage RAW264.7 cells were cultured in high-glucose DMEM complete medium containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% heat-inactivated fetal bovine serum at 37°C with 5% CO2 in 10cm culture dishes. When cells reached 80%–90% confluence, they were passaged. The original medium was discarded, and the cells were washed twice with 4 mL of PBS solution. Then, 1 mL of 0.25% trypsin digest containing EDTA was added to digest the cells for 2 min. Three times the volume of DMEM complete medium was added to terminate the digestion. The cells were gently pipetted repeatedly to suspend them, and collected into 15 mL centrifuge tubes. The cells were centrifuged at 1000 rpm for 4 min at room temperature, and the supernatant was discarded. An appropriate volume of complete culture medium was added and the cells were thoroughly mixed. 10 μL of the cell suspension was aspirated, and 10 μL of trypan blue staining solution was added for viable cell counting. Cells were then passaged at a 1:4 ratio.

[0034] HUVECs cell culture and passage: HUVECs were cultured in high-glucose DMEM complete medium containing 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% heat-inactivated fetal bovine serum at 37°C with 5% CO2 in 10cm culture dishes. When cells reached 80%–90% confluence, they were passaged. The original medium was discarded, and the cells were washed twice with 4 mL of PBS. Then, 1 mL of 0.25% trypsin digest containing EDTA was added to digest the cells for 1 min. After stopping the digestion, three volumes of DMEM complete medium were added to suspend the cells. The cells were then gently pipetted repeatedly to resuspend them. Cells were collected into 15 mL centrifuge tubes and centrifuged at 1000 rpm for 4 min at room temperature. The supernatant was discarded, and an appropriate volume of complete culture medium was added and the cells were thoroughly mixed. 10 μL of the cell suspension was aspirated, and 10 μL of trypan blue staining solution was added for viable cell counting. Cells were then passaged at a 1:4 ratio.

[0035] Experiment on the effects of musk-containing pills and their main components on the viability of RAW264.7 cells: RAW264.7 cells in logarithmic growth phase were harvested, digested, resuspended, and counted using a cell counter at a ratio of 2 × 10⁻⁶ cells / cells. 5 The cells were seeded at a density of 0.1 mL / well in 96-well plates and cultured for 24 hours. The original culture medium was then discarded. 40 mL of complete culture medium was transferred to a 50 mL centrifuge tube, and 8 μL of 1 mg / mL LPS stock solution was added to prepare a working LPS solution with a final concentration of 200 ng / mL. For the drug administration group, 140 μL / well of the drug diluted with LPS working solution was added; for the model group, 140 μL / well of LPS working solution was added; for the blank control group, 140 μL / well of DMEM complete culture medium was added; and for the positive control group, 140 μL / well of dexamethasone solution diluted to 1.276 μM with LPS working solution was added. After incubation for 24 hours, the secretion of nitrite in the culture supernatant was detected using the Griess method according to the nitric oxide assay kit instructions. Standards were diluted with DMEM complete medium to obtain solutions with gradient concentrations of 0, 1, 2.5, 5, 10, 20, 40, 60, and 100 μM for the preparation of standard curves. Standards and samples were added to 96-well plates at 50 μL / well; Griess Reagent I (at room temperature) was added to each well at 50 μL / well; then, Griess Reagent II (at room temperature) was added to each well at 50 μL / well. The absorbance was measured using a multi-plate reader at 540 nm in triplicate. Anti-inflammatory activity was calculated using the following formula, and the results were plotted.

[0036] Experiment on the effect of the main components of Musk Tongxin Dripping Pills on the viability of HUVECs: HUVECs in logarithmic growth phase were harvested, digested, resuspended, and counted using a cell counter at a ratio of 8 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 0.1 mL / well in 96-well plates and cultured normally for 24 hours. The original culture medium was then discarded. For the drug treatment group, 100 μL / well was added to DMEM complete medium to dilute the drug, while for the control group, 100 μL / well was added to DMEM complete medium. After 24 hours of incubation, following the CCK-8 instructions, 100 μL / well of CCK-8 solution diluted in DMEM complete medium was added to each well, and the cells were incubated in the dark for 2 hours. Absorbance was measured at 450 nm using a multi-mode microplate reader. Three parallel experiments were performed, and cell viability was calculated using the following formula and plotted.

[0037] Screening method for the anti-endothelial cell hypoxia injury effect of the main components of Musk Tongxin Dripping Pills: HUVECs in logarithmic growth phase were harvested, digested, resuspended, and counted using a cell counter at a ratio of 8 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 0.1 mL / well in 96-well plates and cultured normally for 24 hours. The original culture medium was then discarded. For the drug treatment group, 100 μL / well was added to DMEM sugar-free medium; for the model group, 100 μL / well was added to DMEM sugar-free medium; and for the blank control group, 100 μL / well was added to DMEM complete medium. The cells were incubated under hypoxic conditions (95% nitrogen, 5% CO2) for 6 hours. The original medium was then discarded. Following the CCK-8 instruction manual, 100 μL / well was added to a 1:1 mixture of DMEM sugar-free and low-glucose medium diluted with CCK-8 solution. The cells were incubated in the dark for 2 hours. The absorbance was measured at 450 nm using a multi-mode microplate reader. Three parallel experiments were performed, and cell viability was calculated using the following formula. The results were then plotted.

[0038] Data statistical methods: All data are expressed as mean ± standard deviation (mean ± SD), and statistical graphs were generated using GraphPad Prism 8 software. Differences between groups were analyzed using one-way ANOVA and Dunnett's test; p < 0.05 was considered statistically significant.

[0039] Screening for effective anti-inflammatory components: A RAW264.7 mouse mononuclear macrophage inflammation model induced by LPS was used. Nitric oxide release inhibition rate was selected as the detection index. Sixteen monomers were chosen as the main components of Shexiang Tongxin Dripping Pill for pharmacodynamic screening, including bufotoxin ester, tanshinone IIA, salvianolic acid C, muscone, bufotoxin, cholic acid, deoxycholic acid, bufotoxin, ursodeoxycholic acid, deacetylated bufotoxin, far-infrared bufotoxin, taurocholic acid, chenodeoxycholic acid, glycocholic acid, ginsenoside Rg1, and ginsenoside Rk3. The effect of the main components of Shexiang Tongxin Dripping Pill on the survival rate of RAW264.7 cells was detected using the CCK8 assay. The results showed that... Figure 3 As shown, at a concentration of 200 μM, the cell viability of all 14 monomeric components was greater than 80% after 24 hours of treatment with RAW264.7 cells, indicating that at this dose, the 14 monomeric components had no significant toxicity to RAW264.7 cells. Only bufotoxin and ginsenoside Rk3 showed some cytotoxic activity at 200 and 400 μM, but the cell viability was greater than 80% at 100 μM. Therefore, the non-toxic concentrations of each monomeric component will be selected for further screening of anti-inflammatory activity.

[0040] A RAW264.7 mouse mononuclear macrophage inflammation model induced by LPS was used, and the anti-inflammatory activity of 16 monomeric components in Musk Tongxin Dripping Pills was investigated using nitric oxide release inhibition rate as the detection index. Results are as follows: Figure 4 As shown, at a concentration of 50 μM, bufotoxin, salvianolic acid C, and ginsenoside Rk3 all significantly inhibited nitric oxide release from RAW264.7 cells, with inhibition rates all greater than 60%, which was higher than that of the positive control drug. This suggests that bufotoxin, salvianolic acid C, and ginsenoside Rk3 may be the active pharmaceutical ingredients responsible for the anti-inflammatory effects of Shexiang Tongxin Dripping Pills in cardiovascular diseases.

[0041] The above experiments can simulate the corresponding intake levels in the human body.

[0042] Therefore, the following anti-inflammatory standards can be established: Screening of effective components for combating hypoxia-induced damage: Sixteen monomers were selected as the main components of Musk Tongxin Dripping Pills for pharmacodynamic screening, including bufotoxin ester, tanshinone IIA, salvianolic acid C, muscone, bufotoxin, cholic acid, deoxycholic acid, bufotoxin, ursodeoxycholic acid, deacetylated bufotoxin, far-infrared bufotoxin, taurocholic acid, chenodeoxycholic acid, glycocholic acid, ginsenoside Rg1, and ginsenoside Rk3. The effects of these 16 monomers on the survival rate of HUVECs cells were detected using the CCK-8 assay. The results are as follows: Figure 5As shown, at a concentration of 200 μM, the cell viability of all 15 monomeric components was greater than 80% after 6 hours of treatment with HUVECs, indicating that at this dose, the 15 monomeric components had no significant toxicity to HUVECs. Only ginsenoside Rk3 showed some cytotoxicity at 200 μM, but the cell viability was greater than 80% at 100 μM. Therefore, the non-toxic concentrations of each monomeric component were subsequently selected to evaluate its anti-endothelial hypoxia injury effect. Using the HUVECs umbilical vein endothelial cell OGD injury model, and with cell viability as the detection index, the effects of 16 monomeric components in Musk Tongxin Dripping Pills on the survival rate of hypoxic-injured HUVECs were investigated. The results are as follows. Figure 6 and Figure 7 As shown, compared with the normal culture group, the cell survival rate of the model group was approximately 40%, indicating that the OGD injury model was successfully established. Compared with the model group, deoxycholic acid and ginsenoside Rg1 at a concentration of 50 μM significantly improved cell survival, and tanshinone C at a low concentration (10 μM) also significantly improved cell survival. This suggests that deoxycholic acid, ginsenoside Rg1, and tanshinone C may be the pharmacodynamic substances that exert the hypoxic injury effect of Shexiang Tongxin Dripping Pills in cardiovascular diseases.

[0043] Based on in vitro studies, salvianolic acid C, which exhibits good anti-inflammatory and anti-hypoxic damage effects, was selected as the main active ingredient in Musk Tongxin Dripping Pills.

[0044] Example 2 Animal experiments on anti-heart failure: The selection and preparation of materials are the same as in Example 1; Drug administration: One week after the operation, based on the ultrasound results, the surviving mice were randomly divided into 3 groups of 6 mice each, namely (1) Sham operation group: TAC operation was performed but ligation was not performed. One week after the model was established, the mice were given 1% sodium carboxymethyl cellulose solution as a solvent control once a day by gavage, each time at 0.1 mL / 10 g. After six weeks of continuous administration, cardiac function was monitored; Chronic heart failure model group (Model): Chronic heart failure model was established according to the method in Chapter 2.2. One week after the model was established, the mice were given 1% sodium carboxymethyl cellulose solution as a solvent control once a day by gavage, each time at 0.1 mL / 10 g. After six weeks of continuous administration, cardiac function was monitored; Tanshinone C administration group (DC): Chronic heart failure model was established according to the conventional method. One week after the model was established, the mice were given Tanshinone C solution as a solvent control once a day by gavage, the dosage was 20 mg / kg per day, the volume of administration was 0.1 mL / 10 g. After six weeks of continuous administration, cardiac function was monitored.

[0045] Echocardiography: Echocardiography monitors cardiac function indices such as CO, LVEF, and LVFS. The specific procedure is as follows: After anesthetizing mice with isoflurane, the hair on the mice's chest is removed using depilatory cream. The mice are then placed supine on the operating table and fixed in a state of anesthesia. The mice continuously inhale isoflurane gas to maintain anesthesia. Coupling gel is applied to the heart area, and an ultrasound probe is used to capture transverse images of the heart. After obtaining continuous and stable images, the mice are removed and returned to their original cages. The mice awaken in approximately 1 minute. The ultrasound images are statistically analyzed using the software accompanying the Vevo 1100 imaging system to obtain indices reflecting cardiac function, such as CO, LVEF, and LVFS, and then plotted.

[0046] echocardiogram results as follows Figure 8 and Figure 9 As shown, in the sham-operated group, the myocardial contraction of mice was normal, and the transverse images of the heart showed large fluctuations; in the model group, the myocardial contraction was weaker, and the transverse images of the heart showed smoother ripples; however, compared with the model group, after six weeks of administration of 20 mg / kg salvianolic acid C, the amplitude of myocardial contraction was significantly improved, and the ripples were larger. Analysis of cardiac function indicators showed that compared with the sham-operated group, the model group had significantly lower levels of cardiac function indicators such as CO, LVEF, and LVFS; while compared with the model group, the salvianolic acid C group had significantly higher levels of cardiac function indicators such as CO, LVEF, and LVFS. This indicates that salvianolic acid C has an effect on improving cardiac function in mice with chronic heart failure, confirming that salvianolic acid C may be one of the main active pharmaceutical ingredients in musk-containing Tongxin dripping pills for treating chronic heart failure.

[0047] Example 3 Based on the verification of Examples 1 and 2, it can be shown that Musk Tongxin Dripping Pills have great potential value in the treatment of cardiovascular diseases. Therefore, based on the results of Examples 1 and 2, the efficacy of Musk Tongxin Dripping Pills is directly verified.

[0048] Preparation of Musk Heart-Clearing Droplet Solution: Grind the dropslet into powder, prepare a solution of the target experimental concentration using DMSO, use sonication to aid dissolution, filter and sterilize using a 0.22μm microporous membrane, and dispense for use.

[0049] The effect of musk-containing Tongxin dripping pills on the survival rate of RAW264.7 cells was detected using the CCK8 assay. The results are as follows: Figure 10 As shown, the cell viability of Musk Tongxin Dripping Pills remained above 80% at concentrations of 50, 100, 200, and 400 μg / mL. Therefore, Musk Tongxin Dripping Pills at concentrations ≤400 μg / mL and below were selected for subsequent anti-inflammatory activity experiments.

[0050] A RAW264.7 mouse mononuclear macrophage inflammation model induced by LPS was used, and the anti-inflammatory activity of musk-containing Tongxin dripping pills was investigated using the inhibition rate of nitric oxide release as the detection index. Results are as follows: Figure 11As shown, Musk Tongxin Dripping Pills, at concentrations of 25–200 μg / mL, exhibited a concentration-dependent inhibitory effect on LPS-induced nitric oxide release in RAW264.7 cells. Furthermore, at a concentration of 200 μg / mL, the inhibition rate of nitric oxide release was greater than 60%, which was higher than that of the positive control group, indicating that Musk Tongxin Dripping Pills have a significant anti-inflammatory effect.

[0051] The effect of musk-containing Tongxin pills on the survival rate of HUVECs was detected using the CCK-8 assay. The results showed that... Figure 12 As shown, at concentrations of 25, 50, and 100 μg / mL, the survival rate of HUVECs cells was above 80%, indicating that at this dosage, the musk-containing Tongxin dripping pills had no significant toxicity to HUVECs cells. Therefore, concentrations of 100 μg / mL and below were selected for subsequent evaluation of their anti-endothelial cell hypoxia injury effect.

[0052] A human umbilical vein endothelial cell (HUVECs) model of OGD injury was used, and cell survival rate was used as the detection index to investigate the effect of musk-containing Tongxin dripping pills on the survival rate of HUVECs under hypoxia injury. Results are as follows: Figure 13 As shown, compared with the normal culture group, the cell survival rate of the model group was about 40%, indicating that the OGD injury model was successfully established. Compared with the model group, Musk Tongxin Dripping Pills at a concentration of 25 μg / mL can significantly improve the survival rate of endothelial cells damaged by OGD, indicating that Musk Tongxin Dripping Pills have an anti-endothelial cell hypoxia injury effect.

[0053] Based on the results of Examples 1-3, it is evident that this invention employs network pharmacology methods combined with transcriptome sequencing technology to conduct in-depth research on the mechanism of action of Musk Tongxin Dripping Pills against cardiovascular diseases, and has carried out preliminary research on its pharmacologically active substances, providing a scientific basis for the clinical application of Musk Tongxin Dripping Pills.

[0054] This invention further confirmed the anti-myocardial infarction and heart failure effects of Shexiang Tongxin Dripping Pills using rat AMI and mouse chronic heart failure models. Results showed that in the rat AMI model, Shexiang Tongxin Dripping Pills significantly reduced the infarct area, decreased cardiomyocyte apoptosis, and alleviated pathological changes such as cardiomyocyte shrinkage and vacuolation. This indicates that the components of Shexiang Tongxin Dripping Pills can improve the blood supply and metabolic environment of cardiomyocytes, reduce cardiomyocyte necrosis caused by ischemia and hypoxia, thereby limiting the expansion of the infarct area. Furthermore, it suggests a possible link to the regulation of apoptosis-related gene expression, such as upregulating the expression of the anti-apoptotic gene Bcl-2 and downregulating the expression of the pro-apoptotic gene Bax, thereby inhibiting the apoptosis process of cardiomyocytes and maintaining the relative stability of cardiomyocyte numbers. In addition, it also has a certain protective effect on cardiomyocytes, maintaining cell cell density. It can restore the normal morphology and function of cells and reduce cell structure damage caused by factors such as ischemia-reperfusion. In a mouse model of chronic heart failure, Musk Tongxin Dripping Pills can significantly improve the cardiac function index and cardiomyocyte hypertrophy, and reduce myocardial fibrosis. This indicates that Musk Tongxin Dripping Pills can enhance the contraction and relaxation function of the myocardium, enabling the heart to pump blood more effectively, thereby improving the overall functional state of the heart. It may prevent excessive growth and hypertrophy of cardiomyocytes by regulating intracellular signaling pathways and gene expression, and reduce the degree of myocardial fibrosis by inhibiting the activation of fibrosis-related signaling pathways, such as the TGF-β / Smad pathway. Further analysis using transcriptome sequencing and network pharmacology techniques revealed that in a rat AMI model, Shexiang Tongxin Dripping Pills significantly restored 63% of the imbalanced genes in the AMI disease network. By regulating the expression of these genes through multiple targets and pathways, it may have reconstructed and stabilized the gene expression network within cardiomyocytes, correcting the pathophysiological changes in the AMI state, thereby exerting its therapeutic effect on AMI. Furthermore, inhibition of apoptosis and the MAPK pathway is one of the important mechanisms by which Shexiang Tongxin Dripping Pills combat AMI. In a mouse chronic heart failure model, Shexiang Tongxin Dripping Pills significantly restored 395 imbalanced genes in the chronic heart failure disease network, with 77.7% of the genes showing a restoration effect greater than 80%. This indicates that Shexiang Tongxin Dripping Pills have a broad regulatory effect on the expression of genes related to chronic heart failure, enabling a large number of abnormally expressed genes to return to near-normal levels. Moreover, inhibition of the TGF-β and MAPK pathways is one of the important mechanisms by which Shexiang Tongxin Dripping Pills combat chronic heart failure. Furthermore, combining transcriptome sequencing and network pharmacology analysis results, this paper further investigated the inflammatory response and vascular endothelial cell damage that play a key role in the occurrence and development of myocardial infarction and heart failure. Using the RAW264.7 mononuclear macrophage inflammation model and the HUVECs umbilical vein endothelial cell hypoxia injury model, the main active substances of Shexiang Tongxin Dripping Pills in the fight against cardiovascular diseases were screened, and in vivo validation studies were conducted.The results showed that bufotoxin, salvianolic acid C, and ginsenoside Rk3 may be the main active substances that exert anti-inflammatory effects in Shexiang Tongxin Dripping Pills in cardiovascular diseases, while deoxycholic acid, ginsenoside Rg1, and salvianolic acid C may be the main active substances that exert anti-endothelial cell hypoxia damage in Shexiang Tongxin Dripping Pills in cardiovascular diseases. Furthermore, salvianolic acid C has a significant anti-chronic heart failure effect in mice.

[0055] In summary, Musk Tongxin Dripping Pills have significant anti-AMI and chronic heart failure effects, and exhibit a significant retrograde effect on the cardiovascular disease network. Inhibition of MAPK, TGF-β, or apoptosis-related pathways is an important mechanism for its anti-cardiovascular disease effects. Furthermore, ginsenoside Rk3, ginsenoside Rg1, salvianolic acid C, and bufotoxin ligand may be important pharmacologically active substances in its anti-cardiovascular disease effects, while salvianolic acid C may be one of the key pharmacologically active substances in its anti-chronic heart failure effects.

[0056] Therefore, based on the above, in addition to being used directly to treat and / or alleviate cardiovascular diseases, Musk Tongxin Dripping Pills can also be used to prepare corresponding drugs and / or health foods to achieve the corresponding effects.

Claims

1. Application of Musk Tongxin Dripping Pills in the Treatment of Cardiovascular Diseases.

2. The application of the Musk Heart-Clearing Drops according to claim 1 in the treatment of cardiovascular diseases, characterized in that... The cardiovascular diseases mentioned include inflammation and / or cellular hypoxia-induced damage caused by cardiovascular lesions and / or injuries, and / or chronic heart failure.

3. The application of the Musk Heart-Clearing Drops according to claim 1 in the treatment of cardiovascular diseases, characterized in that, The treatment of cardiovascular disease includes inhibiting the release of endogenous nitric oxide.

4. The application of the Musk Heart-Clearing Drops according to claim 1 in the treatment of cardiovascular diseases, characterized in that, The treatment of cardiovascular disease includes improving the survival rate of HUVECs cells.

5. The application of the Musk Heart-Clearing Drops according to claim 1 in the treatment of cardiovascular diseases, characterized in that, The treatment of cardiovascular diseases includes improving cardiac function indicators; The cardiac function indicators include CO and / or LVEF and / or LVF.

6. A medicine containing musk-containing Tongxin dripping pills, characterized in that, The drug contains raw materials including musk-containing Tongxin pills; The drug has the function of treating cardiovascular diseases; The treatment of cardiovascular diseases includes inhibiting the release of endogenous nitric oxide and / or increasing the survival rate of HUVECs cells and / or improving cardiac function indicators; The cardiac function indicators include CO and / or LVEF and / or LVF.

7. The drug according to claim 6, characterized in that, The drug also includes one or more other pharmaceutically acceptable carriers and / or excipients.

8. The drug according to claim 6, characterized in that, The drug in question is a commonly used pharmaceutical preparation in pharmaceutics.

9. The medicament according to claim 8, characterized in that, The pharmaceutical preparation is a tablet and / or capsule and / or granule and / or powder and / or liquid preparation.

10. The medicament according to claim 6, characterized in that, The drug is administered orally and / or by injection.

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