Application of traditional Chinese medicine composition for promoting blood circulation to remove blood stasis

Abstract

The invention provides application of a traditional Chinese medicine composition for promoting blood circulation to remove blood stasis. Specifically, the invention provides application of the traditional Chinese medicine composition for promoting blood circulation to remove blood stasis in preparation of medicines for treating coronary artery microvascular diseases and myocardial ischemia and anoxia, myocardial micro-infarction or myocarditis injury caused by the coronary artery microvascular diseases. The traditional Chinese medicine composition for promoting blood circulation to remove blood stasis is prepared from the following components in parts by weight: 300 to 400 parts of radix salviae miltiorrhizae, 150 to 200 parts of radix paeoniae rubra, 150 to 200 parts of rhizoma chuanxiong, 150 to 200 parts of flos carthami and 110 to 130 parts of lignum dalbergiae odoriferae. The invention provides application of the traditional Chinese medicine composition for promoting blood circulation to remove blood stasis in prevention and treatment of coronary artery microvascular diseases and an animal experiment method, and provides a new thought for clinical treatment of coronary artery microvascular diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the present application relates to a kind of application of traditional Chinese medicine composition of promoting blood circulation to remove blood stasis. BACKGROUND

[0002] Coronary microvascular disease (CMVD) refers to the clinical syndrome of exertional angina or objective evidence of myocardial ischemia caused by structural and / or functional abnormalities of the coronary arterioles under the action of various pathogenic factors. It has a high incidence and is one of the important mechanisms leading to myocardial ischemia in coronary heart disease. A large number of research results show that coronary microvascular disease is closely related to adverse cardiovascular events. Therefore, improving coronary microvascular disease has become a hot spot in the prevention and treatment of coronary heart disease. At present, the understanding of the pathogenesis of CMVD is not uniform, which may involve multiple aspects such as microvascular structural and functional abnormalities and extravascular factors (Chinese Journal of Evidence-Based Cardiovascular Medicine, 2020, 12(01): 126-128).

[0003] The treatment goal of modern medicine for CMVD is mainly to control the risk factors of atherosclerosis, improve microcirculation and relieve angina symptoms. The drugs mainly include renin-angiotensin-aldosterone system (RASS) inhibitors, angiotensin-converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB), beta blockers, statins, calcium channel blockers, and nitrate drugs. Previous studies have shown that the current treatment regimen still faces problems such as poor compliance, unstable efficacy and lack of sufficient evidence-based evidence. Therefore, the treatment of coronary microvascular disease is a big difficulty in the field of cardiovascular medicine. Traditional Chinese medicine has the characteristics of multi-level, multi-target and overall treatment. Studies have shown that traditional Chinese medicine treatment based on "promoting blood circulation to remove blood stasis", combined with "regulating qi, eliminating phlegm and strengthening the body", etc. shows certain advantages in improving the prognosis of CMVD.

[0004] CMVD patients often have typical angina symptoms, which belong to the category of "chest pain" in traditional Chinese medicine. The core pathogenesis is caused by different pathological factors leading to heart and blood stasis. The traditional Chinese medicine compound composed of Danshen, Jiangxiang, Chuanxiong, Honghua and Chishao has the effects of promoting blood circulation to remove blood stasis and promoting blood circulation to remove blood stasis. The traditional Chinese medicine compound composed of Danshen, Jiangxiang, Chuanxiong, Honghua and Chishao is currently mainly used for the treatment of coronary atherosclerotic heart disease and has good efficacy. However, its use in CVMD has not been reported. SUMMARY

[0005] The purpose of the present application is to provide an application of a traditional Chinese medicine composition for promoting blood circulation to remove blood stasis.

[0006] To achieve the above object, the application provides application of a traditional Chinese medicine composition for promoting blood circulation and removing blood stasis in preparation of a medicine for treating coronary microvascular disease and myocardial ischemia and hypoxia, myocardial micro-infarction or myocardial injury caused by the coronary microvascular disease.

[0007] According to some specific embodiments of the application, the traditional Chinese medicine composition for promoting blood circulation and removing blood stasis comprises the following ingredients by weight: 351 parts of Danshen, 175 parts of Chishao, 175 parts of Chuanxiong, 175 parts of Honghua and 117 parts of Jiangxiang.

[0008] According to some specific embodiments of the application, the traditional Chinese medicine composition for promoting blood circulation and removing blood stasis can further comprise pharmaceutically acceptable adjuvants such as sucrose and dextrin.

[0009] According to some specific embodiments of the application, the coronary microvascular disease is a coronary microcirculation disorder disease caused by coronary microvascular thrombus blockage.

[0010] According to some specific embodiments of the application, the coronary microvascular thrombus is a coronary microvascular thrombus formed due to inflammation and platelet activation and aggregation induced after endothelial injury of the coronary microvessels.

[0011] According to some specific embodiments of the application, the traditional Chinese medicine composition for promoting blood circulation and removing blood stasis is a traditional Chinese medicine granule.

[0012] The traditional Chinese medicine composition for promoting blood circulation and removing blood stasis of the application can be prepared by referring to the following method comprising the following steps:

[0013] Except for Honghua, the other four ingredients of Danshen and the like are decocted with water for three times, 2 hours for the first time, 1.5 hours for the second time and 1 hour for the third time, the decocted liquid is combined and filtered; Honghua is immersed with water at 80℃ for two times, 2 hours for the first time and 1 hour for the second time, the immersion liquid is combined and filtered, combined with the above-mentioned filtered liquid, concentrated to a thick paste, dried at 80℃, crushed into fine powder, and then sucrose and dextrin are added and mixed, granulated, dried and prepared into 1000g.

[0014] According to some specific embodiments of the application, the method comprises:

[0015] Except for Honghua, the other four ingredients of Danshen and the like are decocted with water for three times, 2 hours for the first time, 1.5 hours for the second time and 1 hour for the third time, the decocted liquid is combined and filtered; Honghua is immersed with water at 80℃ for two times, 2 hours for the first time and 1 hour for the second time, the immersion liquid is combined and filtered, combined with the above-mentioned filtered liquid, concentrated to a thick paste, dried at 80℃, crushed into fine powder, and then sucrose and dextrin are added and mixed, granulated, dried and prepared into 1000g.

[0016] In summary, the application provides an application of a traditional Chinese medicine composition for activating blood and resolving stasis.

[0017] The traditional Chinese medicine composition for activating blood and resolving stasis composed of salvia miltiorrhiza, lycium ruthenicifolium, ligusticum chuanxiong, safflower and red peony root can effectively inhibit platelet activation and aggregation, improve myocardial ischemia and hypoxia, and protect vascular endothelium, thereby reducing coronary microthrombosis, reducing inflammatory infiltration, reducing myocardial microinfarction area, and improving heart function, providing an application of the traditional Chinese medicine composition for activating blood and resolving stasis in preventing and treating coronary microvascular diseases and an animal experiment method, and providing a new idea for clinical treatment of coronary microvascular diseases. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a schematic diagram of left ventricular ejection fraction and myocardial microinfarction area.

[0019] Figure 2 It is a schematic diagram of myocardial pathological staining of a coronary microthrombosis rat model.

[0020] Figure 3 It is a schematic diagram of coronary microthrombosis and myocardial inflammatory infiltration of a rat, wherein rat myocardial HE staining (*200, scale bar=100 μm), n=4; A: Sham group; B: Model group; C: Clo group; D: TM group; E: TH group.

[0021] Figure 4 It is a schematic diagram of myocardial fiber necrosis of a rat, wherein rat myocardial HBFP staining (*100, scale bar=200 μm), n=4; A: Sham group; B: Model group; C: Clo group; D: TM group; E: TH group.

[0022] Figure 5 It is a schematic diagram of rat plasma cTnT level, wherein ns: P≥0.05; *P<0.05; **P<0.01; ***P<0.001.

[0023] Figure 6 It is a schematic diagram of rat plasma ET-1 level, wherein ns: P≥0.05; *P<0.05; **P<0.01; ***P<0.001.

[0024] Figure 7 It is a schematic diagram of rat plasma NO level, wherein ns: P≥0.05; *P<0.05; **P<0.01; ***P<0.001.

[0025] Figure 8 and Figure 9The figures are respectively the rat plasma PF4 and β-TG level diagrams, wherein ns: P≥0.05; *P<0.05; **P<0.01; ***P<0.001.

[0026] Figure 10 The figure is the rat plasma cAMP level diagram, wherein ns: P≥0.05; *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0027] The following detailed description of the implementation process and the beneficial effects of the present application is intended to help the reader better understand the essence and characteristics of the present application, and is not intended to limit the scope of the present application.

[0028] Preparation Example

[0029] Preparation of the blood-activating and stasis-removing traditional Chinese medicine composition: Danshen 351g, Chishao 175g, Chuanxiong 175g, Honghua 175g, Jiangxiang 117g per serving. Except for Honghua, the other four Danshen and the like are decocted with water for three times, 2 hours for the first time, 1.5 hours for the second time, and 1 hour for the third time, and the decoction is combined and filtered. Honghua is immersed with water for two times at 80℃, 2 hours for the first time and 1 hour for the second time, and the immersion liquid is combined and filtered. The combined filtrate is concentrated into a thick paste, dried at 80℃, and pulverized into fine powder. Appropriate amounts of sucrose and dextrin are added and mixed to prepare granules, dried, and prepared into 1000g, thereby obtaining the granules.

[0030] Test Example

[0031] Animal Experiment

[0032] 1. Experimental animals

[0033] 120 SPF healthy Sprague-Dawley (SD) male rats, weighing 200±20g, were used for the experiment after a one-week quarantine period and adaptive feeding with basic animal feed in the animal room.

[0034] 2. Experimental reagents

[0035] (1) Animal administration:

[0036] The blood-activating and stasis-removing traditional Chinese medicine composition (referred to as compound granules) prepared in the preparation example of the present application.

[0037] Clopidogrel hydrogen sulfate tablets (Plavix): purchased from Sanofi-Aventis (Hangzhou) Pharmaceutical Co., Ltd., with the State Drug Standard Code J20130083;

[0038] (2) Modeling:

[0039] Sodium laurate, with a purity of 99%-100%, was purchased from Sigma Company, USA, with CAS: 629-25-4.

[0040] Pentobarbital sodium: purchased from Merk, Germany, CAS: 57-33-0;

[0041] PI3K inhibitor LY294002 (HY-10108) was purchased from MCE, USA;

[0042] 100% dimethyl sulfoxide (DMSO) was purchased from Solarbio, China;

[0043] (3) Pathological morphology:

[0044] 4% paraformaldehyde solution, 500 ml / bottle, purchased from Solarbio, China;

[0045] Absolute ethanol, xylene, hydrochloric acid, ammonia water, etc. required for hematoxylin-eosin staining (HE staining) were purchased from Sinopharm Chemical Reagent Co., Ltd. Hematoxylin staining solution, eosin staining solution and environmentally friendly neutral gum were purchased from Beijing Zhikeyuanbang Biological Technology Co., Ltd.;

[0046] Leagene hematoxylin staining solution, basic fuchsin staining solution, PA decolorizing solution, and HBFP dehydrating agent required for hematoxylin-basic fuchsin-picric acid staining (HBFP staining) were purchased from Genmed Scientific Inc, USA;

[0047] (4) Enzyme-linked immunosorbent assay (ELISA) detection:

[0048] The rat ELISA detection kit included cyclic adenosine monophosphate (cAMP), endothelin 1 (ET-1), cardiac-specific troponin T (cTnT), nitric oxide (NO), platelet factor 4 (PF4), and β-thromboglobulin (β-TG), all of which were purchased from Beijing Dongge Weiyehui Technology Co., Ltd.;

[0049] (5) Heart ultrasound:

[0050] Medical ultrasound coupling agent was purchased from Tianjin Jinyate Science and Technology Development Co., Ltd.

[0051] 3. Experimental equipment

[0052] Electronic balance (model BS224S) was a product of Sartouris, Germany;

[0053] Small animal respirator (model HX-300S) was a product of Chengdu Tailian Software Co., Ltd.;

[0054] Normal temperature high-speed centrifuge (model 5425) was a product of eppendorf, Germany;

[0055] Low speed centrifuge (Model Thermo Micro 17R) is a product of Thermo Fisher Corporation of the United States;

[0056] Cold light source (Model 76053) is a product of Shenzhen Ruivode Life Science and Technology Co., Ltd.;

[0057] Constant temperature water bath (Model TDA-8002) is a product of Beijing Zhonghuan Experimental Furnace Co., Ltd.;

[0058] Pathological section machine (Model RM2235) is a product of Thermo Fisher Corporation of the United States;

[0059] Baking machine (Model DB-B2) is a product of Thermo Fisher Corporation of the United States;

[0060] Optical microscope (Model XSP-9CA) is a product of Shanghai Optical Instrument Factory;

[0061] Enzyme label instrument (Model Thermo FC) is a product of Thermo Fisher Corporation of the United States;

[0062] Shaking mixer is a product of Vortex Corporation of the United States;

[0063] Constant temperature heating pad is a product of Shijiazhuang Bai Rong Electrical Appliance Co., Ltd.;

[0064] Consumables required for modeling and material taking: Gillette blade, straight scissors (large / small), hemostatic forceps, tooth forceps, ophthalmic curved forceps each 4, thoracotome 1, artery clamp 3, needle holder 2; Disposable sterilized tracheal cannula and carotid cannula; Disposable sterilized suture needle with line (4-0) 10 boxes; Disposable sterilized syringe (1ml, 5ml, 10ml) each 10; Pipette (2.5, 20, 100, 200, 1000ml) each one, several different size gun heads; EDTA anticoagulant blood collection tube, 1:9 sodium citrate anticoagulant blood collection tube; 75% alcohol, iodophor, physiological saline, sterile gauze, cotton ball, cotton swab, medical adhesive tape, mask, disposable sterile gloves, liquid nitrogen.

[0065] 4. Experimental method

[0066] (1) Animal grouping and modeling

[0067] ① Animal grouping

[0068] The rats were randomly divided into 7 groups on the day of grouping: sham operation group (Sham), model group (Model), positive control group (Clo), medium-dose blood-activating and stasis-removing traditional Chinese medicine composition group (TM), high-dose blood-activating and stasis-removing traditional Chinese medicine composition group (TH), inhibitor group (Inh), blood-activating and stasis-removing traditional Chinese medicine composition (medium dose) + inhibitor group (Inh-T), 15 rats in each group. After a week of quarantine period and adaptive feeding of basic feed in the animal room, based on different drug intervention of rats in each group (Table 1, the blood-activating and stasis-removing traditional Chinese medicine composition of the application in Table 1 is referred to as "compound granules"), the coronary microthrombosis model was constructed after continuous administration for 4 weeks.

[0069] The drug dose for gavage of rats was calculated according to the recommended daily dose for adults (clopidogrel was 75 mg / day, and the blood-activating and stasis-removing traditional Chinese medicine composition compound granules were 930 mg / day) according to the human-rat body surface area coefficient, and the formula was referred to the fourth edition of "Pharmacological Experimental Methodology". Clopidogrel and medium-high dose blood-activating and stasis-removing traditional Chinese medicine composition were dissolved in distilled water at 0.675 mg / ml, 8.36 mg / ml and 16.72 mg / ml, respectively. The body weight of rats was recorded once a week and the drug dosage was adjusted accordingly. LY294002 was dissolved in 100% DMSO and diluted with corn oil to 0.5 mg / ml for intraperitoneal injection.

[0070] Table 1 Grouping of rats and intervention method

[0071]

[0072] ②Preparation of rat coronary microthrombosis model

[0073] Laboratory site and surgical instruments and articles are sterilized to ensure aseptic operation. The rats are anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 4 ml / kg. The anesthetized rats are fixed on a mouse plate in a supine position. The fur on the front chest of the rat is scraped off and disinfected with iodophor. The modified disposable tracheal tube is inserted into the airway through the oral cavity. After successful insertion, it is fixed, connected to a small animal respirator, and adjusted to the appropriate tidal volume (3 ml / 100 g) and respiratory rate (respiratory ratio 1:1, respiratory rate 88 times / minute). After waiting for the rat to breathe stably, the skin is cut longitudinally along the midline for about 3 cm. The subcutaneous muscle is bluntly separated to the joint connection of the 3rd-4th rib, exposed, clamped and cut off, and the thoracic cavity is opened with a small animal thoracotomy device. The rat thymus is bluntly separated and the pericardium is torn to expose the heart. The ascending aorta is confirmed and separated. After clamping the ascending aorta with a sleeve pipe, a 1 ml syringe is quickly inserted into the left ventricle from the apex. After observing the backflow of the syringe, 0.2 ml of sodium laurate solution (Model group, Clo group, TM group, TH group, Inh-T group, Inh group) or normal saline (Sham group) with a concentration of 1 g / L is injected into the ventricular cavity. After maintaining the syringe state for 30 seconds, the aorta is opened, the 1 ml syringe is removed, and the heart is reset. The thoracic cavity is flushed with 37°C normal saline. After the heart rate stabilizes, the thoracic cavity is closed layer by layer and sutured. The surgical incision is disinfected again with iodophor. After the rat's spontaneous breathing is stable, the tracheal tube is removed and placed on a 35°C constant temperature heating pad to help it recover body temperature.

[0074] 5. Index detection

[0075] (1) Detection purpose and time point

[0076] ① Histopathological observation: 24 hours after successful modeling, the heart is removed, and HE and HBFP staining are performed on the coronal section to observe the histopathological morphology and infarction area.

[0077] ② Platelet function detection: 24 hours after successful modeling, the rat is anesthetized, and blood is taken from the abdominal aorta. The plasma PF4, β-TG, and cAMP levels are detected by enzyme-linked immunosorbent assay.

[0078] ③ Vascular endothelial function detection: 24 hours after successful modeling, the rat is anesthetized, and blood is taken from the abdominal aorta. The plasma NO and ET-1 levels are detected by enzyme-linked immunosorbent assay.

[0079] ④ Myocardial injury detection: 24 hours after successful modeling, the rat is anesthetized, and the left ventricular ejection fraction (LVEF) is measured using a small animal ultrasound machine. 24 hours after successful modeling, the rat is anesthetized, and blood is taken from the abdominal aorta. The plasma cTnT level is detected by enzyme-linked immunosorbent assay.

[0080] (2) Histological staining

[0081] Each group randomly selected 6 rats, 24 hours after the success of the model, before the heart stopped beating, take off the whole heart into the physiological saline, make it pump out the residual blood in the ventricular cavity. Select the left ventricular segment papillary muscle level specimen, fixed in 4% paraformaldehyde solution, coronal section for the production of HE staining and HBFP staining. Case staining section placed under the optical microscope (x40) observation and scanning into images, where HE staining for observation of myocardial tissue structure, microthrombus and inflammatory infiltration; HBFP staining to observe myocardial injury, by Image-Pro-Plus software to calculate the target color area accounted for the proportion of myocardial total area, that is, the proportion of myocardial microinfarction injury area, expressed in percentage (%).

[0082] (3) Rat heart ultrasound

[0083] Each group randomly selected 6 rats, 24 hours after the success of the model, the rat was shaved in the front chest, pre-anesthetized with isoflurane inhalation, supine position fixed on the small animal ECG monitoring board, continuous inhalation anesthesia, so that the heart rate of the rat was stable at 380-410 times. The B-type and M-type echocardiogram of the rat parasternal left ventricular long axis section and parasternal short axis papillary muscle section were detected by small animal ultrasound machine, and the left ventricular ejection fraction (LVEF) of the rat was measured.

[0084] (4) ELISA

[0085] After 24 hours of successful modeling, the abdominal aorta was taken blood and injected into an anticoagulant tube containing sodium citrate, mixed well, centrifuged at 1000 r / min for 10 minutes, and the upper clear liquid was taken as platelet-rich plasma; centrifuged at 3500 r / min for 10 minutes, and the upper clear liquid was taken. The contents of NO, ET-1, cTnT, cAMP, PF4 and β-TG in the plasma of each group of rats were detected by enzyme-linked immunosorbent assay, and the specific steps were as follows according to the instructions of the kit:

[0086] ① Sample preparation: 50 μl of platelet-rich plasma was diluted with 150 μl of calcium-free table solution, centrifuged at 3000 r / min for 10 minutes, the supernatant was discarded, 200 μl of calcium-free table solution was added for washing once, the remaining platelet pellet was resuspended with 100 μl of calcium-free table solution, 1 μl of ADP (final concentration 20 μmol / L) was added, incubated at room temperature for 5 minutes, then 100 μl of 0.1 mol / L hydrochloric acid was added, incubated on ice for 10 minutes to terminate the reaction, then centrifuged at 4°C, 9000 r / min for 10 minutes, and the supernatant was extracted for ELISA detection.

[0087] ②Standard dilution: Prepare 7 EP tubes, labeled as No. 1-7. Add 100 μl of the original concentration standard to No. 1 tube, and add 100 μl of the standard dilution to No. 2-7 tubes. Add 100 μl of the original concentration standard to No. 2 tube, mix well, and then use a pipette to take out 100 μl of the liquid and add to No. 3 tube, mix well, and then take out 100 μl of the liquid and add to No. 4 tube, and so on until No. 7 tube. Finally, the dilution concentrations of the standards in No. 1-7 tubes are: 240 nmol / L, 120 nmol / L, 60 nmol / L, 30 nmol / L, 15 nmol / L, 7.5 nmol / L, and 3.75 nmol / L, respectively.

[0088] ③Add antibody: Take out the enzyme-labeled coated plate, wash with plate washing solution, and shake dry for 3 times. Set a blank well and add 50 μl of the matching solvent, and add 50 μl of the monoclonal antibody provided in the kit to the rest of the wells, seal the plate, and then incubate on a shaker at 500 r / min for 1 hour.

[0089] ④Add sample: Wash the plate with plate washing solution for 4 times, and shake dry. Add 50 μl of the conjugate provided in the kit to each well, then add 100 μl of the standard of different concentrations to the standard wells in turn, add 100 μl of the sample to the sample wells, add 100 μl of the matching solvent to the blank wells and the control wells, then seal the plate, and then incubate on a shaker at 500 rpm for 2 hours.

[0090] ⑤Color development: After washing the plate for 4 times, add 100 μl of the color developing agent to each well, and incubate at room temperature for 30 minutes.

[0091] ⑥Termination and determination: Add 100 μl of the termination solution to each well to terminate the reaction, and use the enzyme-labeled instrument to determine the absorbance of each well at 450 nm within 15 minutes, with the blank well set to zero, and the control well corrected. Draw the linear regression curve of the standard with the concentration as the horizontal coordinate and the absorbance as the vertical coordinate, and calculate the concentrations of NO, ET-1, cTnT, cAMP, PF4, and β-TG in each sample according to the curve equation.

[0092] 6. Statistical method

[0093] SPSS 20.0 software was used for statistical analysis, and GraphPad Prism 6 software was used for plotting. The data were expressed as mean ± standard deviation (mean ± SD), the comparison among multiple groups of normally distributed data was performed by one-way ANOVA, and the pairwise comparison between groups was performed by LSD test (homogeneous variance) or Games-Howell test (inhomogeneous variance); the comparison among multiple groups of non-normally distributed data was performed by Kruskal-Wallis rank sum test. The hypothesis test was a two-sided test, and the difference was considered statistically significant at P<0.05.

[0094] 7. Experimental results

[0095] (1) Evaluation of coronary microthrombosis model

[0096] After 24 hours of modeling, 6 rats from each of the Sham and Model groups were randomly selected. After isoflurane inhalation anesthesia, the rats were subjected to cardiac ultrasound to observe the left ventricular ejection fraction (LVEF) of the rats. The hearts of the rats were isolated to observe the microthrombosis and myocardial microinfarction area of the rats by HE and HBFP staining. The results are shown in Figure 1 Compared with the rats in the Sham group, the rats in the Model group had a significantly lower LVEF. The HE staining results Figure 2 showed that the rats in the Model group had microthrombosis in the myocardial arterioles (less than 100 μm), endothelial detachment in the microvessels, and inflammatory cell infiltration around the thrombus. The thrombus structure was mainly composed of red blood cells and platelets. The rats in the Sham group had an intact myocardial endothelium and no obvious thrombosis. The HBFP staining results Figure 2 showed that the rats in the Sham group had no obvious necrosis (area ratio <0.5%), and the rats in the Model group had a focal distribution of myocardial fiber necrosis, with a microinfarction area of 6.46%±1.02%. Compared with the rats in the Sham group, the difference was statistically significant (P<0.001), indicating that the rat coronary microthrombosis model was successfully established.

[0097] (2) Improvement of cardiac function

[0098] As shown in Table 2, there was no statistically significant difference in the baseline level of LVEF and heart rate during the ultrasound examination of the rats in each group before modeling (P>0.05). After modeling, there was no statistically significant difference in the heart rate of the rats in each group during the ultrasound examination (P>0.05). The LVEF of the rats treated with clopidogrel and the compound granules was significantly higher than that of the rats in the Model group (P<0.01). The LVEF of the rats in the Clo group was significantly higher than that of the rats in the TM group, but there was no statistically significant difference in the efficacy of clopidogrel and the high-dose compound granules in improving the LVEF of the rats (P>0.05). The LVEF of the rats treated with the blood-activating and stasis-removing traditional Chinese medicine composition + PI3K inhibitor and the rats treated with the PI3K inhibitor alone was significantly higher than that of the rats in the Model group (P<0.01), but there was no obvious difference between the two groups and between the two groups and the TM group (P>0.05).

[0099] Table 2 Heart rate and left ventricular ejection fraction of rats in each group after modeling

[0100]

[0101]

[0102] The data are represented as mean ± standard deviation (mean ± SD), n=6; **P<0.01 Vs. Model; ▲P<0.05 Vs. Clo.

[0103] (3) Reducing coronary microthrombosis

[0104] Compared with the Sham group, coronary microthrombosis and inflammatory cell infiltration of different degrees were observed in the arterioles of other groups under HE staining. Figure 3 Among them, the microthrombosis and inflammatory cells of the Model group were the most obvious, while the microthrombosis and inflammatory cell infiltration of the Clo group, TM group and Th group were improved to different degrees compared with the Model group. Under HBFP staining, focal myocardial infarction of different degrees was observed in the Model group, Clo group, TM group and TH group. Figure 4 Among them, the infarct area ratio of the Clo group and TH group was similar (P>0.05), and the infarct area ratio of the TM group was higher than that of the Clo group with statistical difference (P<0.05); the infarct area ratio of the Clo, TM and TH groups was significantly smaller than that of the Model group, and the difference was statistically significant (P<0.01), as shown in Table 3.

[0105] Table 3 Myocardial microinfarct area of rats in each group after modeling under HBFP staining

[0106]

[0107] Data are expressed as mean ± SD (n=4); **P<0.01 Vs. Model; ▲ P<0.05 Vs. TM.

[0108] (4) Anti-myocardial injury

[0109] As shown in Figure 5 , the plasma cTnT level of the Model group was significantly higher than that of the Sham group (P<0.01), and after the early intervention of clopidogrel and middle-high dose compound granules, the cTnT level of rats decreased to a certain extent (P<0.01), and there was no statistical difference in the effect of reducing plasma cTnT level among the three groups (P>0.05). The co-preconditioning of compound granules and PI3K inhibitor can significantly reduce the plasma cTnT level compared with the Model group (P<0.05); while there was no statistical difference between the Inh group and the Inh-T group (P>0.05).

[0110] (5) Protection of vascular endothelium

[0111] Compared with the Sham group, the plasma ET-1 level of the Model group was significantly increased (P<0.01). Compared with the Model group, the plasma ET-1 levels of the Clo group and the TM group were significantly decreased (P<0.01, P<0.05), and there was no significant difference between the two groups (P>0.05). The plasma ET-1 level of the high-dose compound granule group was not significantly different from that of the Model group (P>0.05), and was significantly higher than that of the Clo group and the TM group (P<0.01). After pretreatment with the PI3K inhibitor + compound granule, the plasma ET-1 level of the rats was significantly lower than that of the Model group (P<0.01) and was not significantly different from that of the Clo group and the TM group (P>0.05). The plasma ET-1 level of the Inh group was not significantly different from that of the Model group, the Inh + T group, and the Clo group (P>0.05). See Figure 6 .

[0112] As shown in Figure 7 , the plasma NO level of the Model group was significantly lower than that of the Sham group (P<0.01). After pretreatment with clopidogrel and high-dose compound granules, the plasma NO level of the rats was increased to a certain extent compared with the Model group and the TM group (P<0.01, P<0.05), and the plasma NO level of the Clo group was significantly higher than that of the TH group (P<0.01). There was no significant difference in the plasma NO level between the TM group and the Model group (P>0.05). Pretreatment with compound granules and PI3K inhibitors significantly increased the plasma NO level compared with the Model group and the TM group (P<0.05).

[0113] (6) Anti-platelet

[0114] As shown in Figure 8 and Figure 9 , compared with the Sham group, the plasma PF4 and β-TG levels of the Model group were significantly increased (P<0.01). After pretreatment with clopidogrel and medium- and high-dose compound granules, the two indicators of the rats were significantly decreased compared with the Model group (P<0.01). Among them, the effect of the high-dose group on reducing plasma PF4 and β-TG and the effect of the medium-dose group on reducing β-TG were not significantly different from that of clopidogrel (P>0.05), but the effect of the medium-dose group on reducing plasma PF4 was weaker than that of clopidogrel (P<0.05). Pretreatment with PI3K inhibitors and compound granules significantly reduced the plasma PF4 and β-TG of rats compared with the TM group (P<0.05), but there was no significant difference compared with the Inh group (P>0.05).

[0115] As shown in Figure 10Compared with the sham group, the plasma cAMP level of the model group was significantly decreased (P<0.01). Compared with the model group, the plasma cAMP levels of the Clo group, the TM group, the TH group and the Inh-T group were significantly increased (P<0.05). The effects of the medium and high dose of the compound granules on decreasing the plasma cAMP level were weaker than that of clopidogrel (P<0.05), and the effect of the medium dose of the compound granules was similar to that of the high dose (P>0.05). Compared with the TM group, the plasma cAMP level of the Inh-T group was significantly decreased (P<0.05), but there was no statistical difference compared with the Inh group (P>0.05).

Claims

1. Application of a traditional Chinese medicine composition for activating blood and removing blood stasis in the preparation of a drug for treating coronary microvascular disease and myocardial ischemia and hypoxia, myocardial microinfarction or myocardial inflammatory damage caused by the coronary microvascular disease.

2. The use according to claim 1, wherein, The traditional Chinese medicine composition for activating blood and removing blood stasis comprises the following components in parts by weight: Danshen 300-400 parts, Chishao 150-200 parts, Chuanxiong 150-200 parts, Honghua 150-200 parts and Jiangxiang 110-130 parts.

3. Use according to claim 1 or 2, wherein, The traditional Chinese medicine composition for activating blood and removing blood stasis comprises the following components in parts by weight: Danshen 351 parts, Chishao 175 parts, Chuanxiong 175 parts, Honghua 175 parts and Jiangxiang 117 parts.

4. Use according to claim 3, wherein, The coronary microvascular disease is a coronary microcirculation disorder disease caused by coronary microvascular thrombosis. The coronary microvascular thrombus is a coronary microvascular thrombus formed by induced inflammation and platelet activation and aggregation after endothelial injury of the coronary microvessels.

Images