Traditional Chinese medicine composition for preventing and treating coronary artery microvascular disease caused by ischemia reperfusion injury as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention belongs to the field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for preventing and treating coronary artery microvascular disease caused by ischemia reperfusion injury and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 10 to 20 parts of rhizoma coptidis with ginger, 9 to 15 parts of rhizoma pinelliae with ginger, 15 to 30 parts of pericarpium trichosanthis, 5 to 15 parts of cassia twig, 5 to 30 parts of raw radix astragali seu hedysari and 15 to 30 parts of radix salviae miltiorrhizae. Animal experiments show that the composition can improve heart blood perfusion, protect capillary structures and barrier functions and reduce myocarditis, thereby improving cardiac dysfunction. Cell experiments prove that the compound can activate endothelial cell mitochondria autophagy, eliminate damaged mitochondria, relieve oxidative stress and cell apoptosis and improve the endothelial function. In conclusion, the composition plays an endothelial protection role by promoting microcirculation perfusion and activating mitochondrial autophagy, and coronary artery microvascular dysfunction caused by ischemia reperfusion injury is effectively prevented and treated.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically relating to a traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury, its preparation method, and its application. Background Technology

[0002] Percutaneous coronary intervention (PCI) is the primary treatment for acute myocardial infarction. However, although reperfusion therapy can effectively salvage dying myocardium, over 50% of patients experience no-reflow after coronary artery recanalization, manifesting as myocardial perfusion impairment. The main pathological basis for this is coronary microvascular dysfunction caused by ischemia / reperfusion injury (I / R injury). Reducing microvascular reperfusion injury is a potential direction for improving the long-term prognosis of myocardial infarction patients, and the prevention and treatment of coronary microvascular disease is gradually becoming a key focus for clinicians.

[0003] I / R damage can lead to the destruction of cardiac microvascular structure and function, manifested as endothelial cell apoptosis, loss of barrier function, increased vascular permeability, and vascular collapse. This, in turn, restricts the delivery of drugs and oxygen to the infarcted area, and is a key factor limiting cardioprotective effects and leading to heart failure. In recent years, mitochondrial dysfunction and autophagy defects have been shown to play important roles in microvascular endothelial cell injury. Mitophagy is one of the key mechanisms for maintaining endothelial homeostasis and energy metabolism, and I / R damage often leads to impairment of the mitophagy pathway, thereby inducing endothelial apoptosis and exacerbating microcirculatory dysfunction.

[0004] Nicorandil is a commonly used drug for treating coronary microangiopathy (CMVD) and is one of the most studied drugs. Nicorandil is an ATP-sensitive potassium channel opener and a nitrate drug. Because it opens mitochondrial KATP channels, it is used in ischemic preconditioning (IPC) to limit myocardial infarction and reduce subsequent severe ischemic damage. In the treatment of coronary microangiopathy, nicorandil effectively dilates microvessels, improves myocardial perfusion, and relieves ischemic symptoms; therefore, it is widely used in secondary prevention and chronic treatment of ischemic heart disease.

[0005] However, the application of nicorandil also has certain limitations. On the one hand, nicorandil may cause side effects such as hypotension, headache, gastrointestinal symptoms, and oral ulcers, making it unsuitable for patients with heart failure. On the other hand, as a single-target drug, nicorandil cannot comprehensively intervene in all pathogenic processes, resulting in relatively limited efficacy. In contrast, traditional Chinese medicine, with its multi-target efficacy and fewer side effects, is receiving increasing attention and importance in the prevention and treatment of coronary microvascular disease. Therefore, developing a traditional Chinese medicine compound preparation that can effectively prevent and treat coronary microvascular disease caused by ischemia-reperfusion injury has significant clinical implications. Summary of the Invention

[0006] The first objective of this invention is to provide a traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury; the second objective of this invention is to provide a method for preparing the traditional Chinese medicine composition; and the third objective of this invention is to provide the application of the traditional Chinese medicine composition.

[0007] According to a first aspect of the present invention, a traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury is provided, wherein the raw materials comprise, by weight, 10-20 parts of Coptis chinensis, 9-15 parts of Pinellia ternata, 15-30 parts of Trichosanthes kirilowii peel, 5-15 parts of Cinnamomum cassia, 5-30 parts of Astragalus membranaceus, and 15-30 parts of Salvia miltiorrhiza.

[0008] This invention posits that coronary microangiopathy following ischemia-reperfusion injury falls under the category of "chest pain and heart pain," characterized by a complex interplay of deficiency and excess. Deficiency is particularly prevalent in Qi deficiency and Yang deficiency; excess is characterized by Qi stagnation, cold coagulation, phlegm turbidity, and blood stasis, which can interact, with blood stasis and phlegm turbidity being the most common. Both the deficiency and excess aspects of this disease are guided by the key pathogenesis of "obstruction of the heart vessels, leading to pain due to obstruction." During acute attacks, the excess aspect predominates, with phlegm turbidity and blood stasis obstructing the flow, leading to heat accumulation. The interplay of phlegm, blood stasis, and heat further obstructs the heart vessels, causing symptoms such as chest fullness and pain, and shortness of breath.

[0009] This formula is based on the TCM diagnostic system of "chest pain and heart pain", with the pathogenesis of "qi deficiency and yang deficiency, phlegm and blood stasis, and obstruction of the heart vessels" and the treatment principle of "tonifying qi and promoting yang, activating blood and resolving phlegm". It is supplemented by tonifying qi and raising yang, warming and promoting heart yang, and activating blood and unblocking the collaterals.

[0010] In this formula, Trichosanthes peel is the chief ingredient, used to soothe the chest, regulate qi, clear heat, and resolve phlegm. Ginger, Coptis chinensis, and Pinellia ternata are the assistant ingredients, used together to clear heat, dry dampness, and relieve nausea and vomiting. Salvia miltiorrhiza, Astragalus membranaceus, and Cinnamomum cassia are the adjuvant ingredients. Salvia miltiorrhiza invigorates blood circulation and removes blood stasis, improving microvascular blood flow stagnation, and works synergistically with Trichosanthes peel to invigorate blood circulation and resolve phlegm. Astragalus membranaceus tonifies qi and raises yang, improving insufficient microcirculation and microvascular tension. Cinnamomum cassia invigorates heart yang, and works synergistically with Astragalus membranaceus to enhance warming, invigorating, and lifting effects. The combination of Salvia miltiorrhiza, Astragalus membranaceus, and Cinnamomum cassia, on the one hand, tonifies qi and raises yang, unblocks the meridians, and relieves pain, improving the deficiency of both qi and yang, which weakens blood circulation; on the other hand, it harmonizes the other herbs, using both warming and cooling properties, and combining tonification with purgation. The entire formula works to soothe the chest, invigorate yang, resolve phlegm, dissipate nodules, and invigorate blood circulation. This approach shares similarities with modern methods for enhancing vasodilation, reducing coronary microvascular obstruction, and protecting endothelial cells to reduce apoptosis.

[0011] The raw materials for this invention are sourced as follows: Turmeric Coptis: Processed form of Coptis chinensis. Coptis chinensis is the dried rhizome of *Coptis chinensis* Franch., *Coptis deltoidea* CYet Hsiao, or *Coptis teeta* Wall., all belonging to the genus *Coptis* of the Ranunculaceae family. The processing method for turmeric coptis is as follows: Wash and crush fresh ginger, add water and press to extract the juice. Add an appropriate amount of water to the ginger residue and press again, combining the juices to obtain "ginger juice." The ratio of ginger juice to fresh ginger is 1:1. Take Coptis slices, mix them with ginger juice, place them in a wok, and heat over low heat until the ginger juice is absorbed and the mixture is dry. Remove and air dry. For every 100 kg of Coptis slices, use 12.5 kg of fresh ginger juice. Coptis clears heat and relieves stagnation; ginger processing helps to reduce the adverse effects of its bitter and cold nature on the stomach.

[0012] Ginger-processed Pinellia: A processed form of Pinellia ternata. Pinellia ternata is the dried tuber of Pinellia ternata (Thunb.) Breit., a plant in the Araceae family. The processing method for ginger-processed Pinellia ternata is as follows: Take clean Pinellia ternata, separate them by size, soak them in water until the inside is no longer dry, then remove them; separately, take sliced ​​fresh ginger, decoct it in water, add alum and boil the Pinellia ternata together until thoroughly cooked, remove, and air dry, or air dry until semi-dry, then dry completely; or slice thinly and dry completely. For every 100kg of clean Pinellia ternata, use 25kg of fresh ginger and 12.5kg of alum. Pinellia ternata resolves phlegm and dissipates nodules; ginger processing helps to reduce the adverse effects of its bitter and cold nature on the stomach.

[0013] Trichosanthes peel: The dried, mature pericarp of Trichosanthes kirilowii Maxim. or T. uniflora Hao., both belonging to the Cucurbitaceae family.

[0014] Cinnamon twig: The dried young twig of Cinnamomum cassia Presl., a plant in the Lauraceae family.

[0015] Raw Astragalus: A processed form of the Chinese medicinal herb Astragalus, which is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., which is cleaned, softened and cut.

[0016] Danshen: The dried root and rhizome of *Salvia miltiorrhiza* Bge., a plant in the Lamiaceae family. Bitter and slightly cold in nature. It enters the Heart and Liver meridians. In some embodiments, the raw material composition by weight includes: 15 parts of Coptis chinensis, 15 parts of Pinellia ternata, 30 parts of Trichosanthes kirilowii peel, 15 parts of Cinnamomum cassia, 30 parts of Astragalus membranaceus, and 15 parts of Salvia miltiorrhiza.

[0017] In some embodiments, pharmaceutically acceptable excipients may also be added. Specifically, the excipients used are carriers or excipients commonly used in the art, including but not limited to starch, lactose, glucose, sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose, malt, gelatin, polyols (such as propylene glycol, glycerol, mannitol), tablets, etc., to help improve the stability or activity of the drug, or to produce an acceptable taste or odor when taken orally.

[0018] In some embodiments, the dosage form of the traditional Chinese medicine composition is a decoction, oral solution, powder of traditional Chinese medicine extract, powder, granules, tablets, pills, or capsules. This facilitates clinical administration.

[0019] According to a second aspect of the present invention, a method for preparing the above-mentioned traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury is provided, comprising the following steps: Add water according to a water-to-medicine ratio of (8-10):1, soak for 30-40 minutes, first decoct over high heat for 30-60 minutes, then simmer over low heat for 1-1.5 hours, stirring the herbs 2-3 times during the decoction. Filter the first decoction, add 8-10 times the weight of water to the dregs again, bring to a boil, then simmer over low heat for 1-1.5 hours. Filter the decoction, combine the two filtrates, and the resulting decoction is ready. The obtained decoction can be further concentrated and dried to produce various dosage forms such as herbal extract powder, powder, granules, tablets, pills, and capsules.

[0020] According to a third aspect of the present invention, the use of the above-described traditional Chinese medicine composition for preventing and treating coronary microangiopathy caused by ischemia-reperfusion injury is provided in the preparation of a medicament for preventing and treating coronary microangiopathy. Specifically, coronary microangiopathy refers to coronary microangiopathy caused by ischemia-reperfusion injury.

[0021] According to a fourth aspect of the present invention, the above-described traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury is provided for use in the preparation of a medicament for preventing and treating structural and functional disorders of coronary microvascular endothelium.

[0022] According to a fifth aspect of the present invention, the application of the above-mentioned traditional Chinese medicine composition for preventing and treating coronary microangiopathy caused by ischemia-reperfusion injury is provided in the preparation of a drug for preventing and treating coronary slow flow syndrome. Coronary slow flow syndrome is an abnormally slow blood flow phenomenon detected by angiography in the absence of interventional treatment, including the absence of coronary artery spasm, thrombosis, dissection, myocardial bridging, or other mechanical obstruction. It is an important manifestation of coronary microangiopathy, and its pathophysiological basis mainly lies in the structural or functional abnormalities of the coronary microcirculation.

[0023] According to a sixth aspect of the present invention, the above-described traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury is provided for use in the preparation of a medicament for preventing and treating myocardial infarction and microvascular heart failure.

[0024] When the traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury is in the form of a decoction, adult patients are given one dose per day, based on a body weight of 60 kg, with each dose being 150-250 mL and the amount of raw herbs being approximately 105 g, to be taken orally in 2-3 divided doses every 6-8 hours.

[0025] When the traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury is in the form of a solid preparation (powder, powder, granules, tablets, pills or capsules of traditional Chinese medicine extract), it is given to adult patients once a day. For adults, the dosage is calculated based on a body weight of 60 kg, and the effective dose of the solid preparation is equivalent to 105g of raw herbs per day. The dose is given every 6 to 8 hours.

[0026] The beneficial effects of this invention include: (1) Animal and cell experiments have shown that the traditional Chinese medicine composition of the present invention has the following pharmacological effects: improving cardiac function and increasing coronary artery blood perfusion; reducing the apoptosis rate of vascular endothelial cells; activating the LC3B and PINK1 / Parkin pathways, promoting mitophagy, and reducing endothelial cell damage; downregulating VCAM-1 expression and alleviating endothelial inflammatory response; increasing VE-Cadherin expression, stabilizing vascular connectivity, and restoring vascular barrier function. This suggests that the traditional Chinese medicine composition of the present invention can effectively prevent and treat coronary microangiopathy, including coronary microangiopathy caused by ischemia-reperfusion injury, coronary microvascular endothelial structural and functional disorders, coronary slow flow syndrome, and myocardial infarction with microvascular dysfunction.

[0027] (2) The traditional Chinese medicine composition of the present invention has a clear targeting effect and can effectively prevent and treat microvascular diseases by improving endothelial mitochondrial autophagy and protecting barrier function. In vitro and in vivo experiments have verified its multi-dimensional improvement effects on cardiac function, microvascular perfusion, inflammation inhibition, and energy metabolism, and it has good clinical application prospects. Attached Figure Description

[0028] Figure 1 These are M-mode ultrasound images and cardiac function data of mice in each group during the animal experiments of this invention.

[0029] Figure 2 This shows the HE staining of mouse heart tissue in each group during the animal experiments of this invention.

[0030] Figure 3 This describes the cardiac blood perfusion status of mice in each group during the animal experiments of this invention.

[0031] Figure 4 This describes the expression of VCAM-1 in the cardiac endothelial cells of mice in each group during the animal experiments of this invention.

[0032] Figure 5 This describes the expression of VE-Cadherin in the cardiac endothelial cells of mice in each group during the animal experiments of this invention.

[0033] Figure 6 This describes the expression of LC3B in the cardiac vascular endothelium of mice in each group during animal experiments of this invention.

[0034] Figure 7 These are the results of cell viability and apoptosis of endothelial cells in each group during the cell experiments of this invention.

[0035] Figure 8 This shows the expression of VE-Cadherin and VCAM-1 proteins in each group of cells during the cell experiments of this invention.

[0036] Figure 9 This describes the mitochondrial damage in each group of cells during the cell experiments of this invention.

[0037] Figure 10 This describes the co-localization of mitochondria and lysosomes in the cells of each group during the cell experiments of this invention.

[0038] Figure 11 This describes the expression of mitochondrial autophagy-related proteins in each group of cells during the cell experiments of this invention. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all raw materials and reagents used in the present invention are commercially available.

[0040] Example 1 The traditional Chinese medicine decoction for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury in this embodiment is prepared by the following steps: (1) Put 15g of Coptis chinensis, 15g of Pinellia ternata, 30g of Trichosanthes kirilowii peel, 15g of Cinnamomum cassia, 30g of Astragalus membranaceus, and 15g of Salvia miltiorrhiza into a medicine pot; (2) Add water according to a water-to-medicine ratio of 9:1, with the liquid level about 5 cm above the surface of the medicine. Soak for about 35 minutes. First, boil over high heat for 50 minutes, then boil over low heat for 1 hour. Stir the medicine about 3 times during the boiling process to prevent the liquid from overflowing, drying out, or burning. When the liquid is 1 / 3 of its original volume, filter the first decoction. Add 1000 mL of water to the dregs again, boil over high heat, and then boil over low heat for 1 hour. When the liquid is about 400 mL, filter the decoction. Combine the two decoctions to obtain the final product.

[0041] Example 2 The preparation method of the traditional Chinese medicine extract powder for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury in this embodiment includes the following steps: (1) Put 75g of Coptis chinensis, 75g of Pinellia ternata, 150g of Trichosanthes kirilowii peel, 75g of Cinnamomum cassia, 150g of Astragalus membranaceus, and 75g of Salvia miltiorrhiza into a medicine pot.

[0042] (2) Add 4800 mL of water according to the water-to-medicine ratio of 8:1, with the liquid level about 3 cm above the surface of the medicine. Soak for 30 minutes, first decoct over high heat for 30 minutes, then simmer over low heat for 1 hour. Stir the medicine 3 times during the decoction process to prevent the liquid from overflowing, drying out, or burning. When 1600 mL of the liquid remains, filter the first decoction into a 5 L conical flask. Add 4800 mL of water to the residue again, bring to a boil over high heat, then simmer over low heat for 1 hour. When 1600 mL of the liquid remains, filter the second decoction and combine it into the conical flask to obtain the decoction.

[0043] (3) The decoction was distilled under reduced pressure using a medium-sized rotary evaporator. The water bath temperature was set to 60℃, the pressure to -0.1MPa, the condenser temperature to -15℃, and the rotation speed to 60 rpm. After the boiling bubbles stabilized, the temperature was gradually increased to 70℃ to concentrate the decoction to about 800mL. The solution was then placed in a 1 L beaker and divided into 6 small bowls. The bowls were then freeze-dried continuously for 7 days at a freeze-drying temperature of -80℃ using a vacuum freeze dryer (Labconco, Freezone) to obtain 193.8g of freeze-dried extract powder. The extraction rate was 193.8g / 600g×100%=32.3%.

[0044] Example 3 The traditional Chinese medicine decoction for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury in this embodiment is prepared by the following steps: (1) Put 20 g of Rhizoma Coptidis Rhizoma Zingiberis Recens, 9 g of Pinelliae Rhizoma Praeparatum Cum Zingibere, 15 g of Fructus Trichosanthis Pericarpium, 10 g of Ramulus Cinnamomi, 20 g of Radix Astragali seu Hedysari, and 20 g of Radix Salviae Miltiorrhizae into a medicine pot; (2) Add water according to a water-to-medicine ratio of 9:1, with the liquid level about 5 cm above the medicine surface. Soak for about 35 min, first decoct with strong fire for 50 min, then switch to slow fire and decoct for 1 h. Stir the medicine materials about 3 times during the decocting process to prevent the liquid medicine from overflowing, drying up, or burning. When the liquid medicine remains 1 / 3 of the original volume, filter to obtain the first decoction. Add 900 mL of water to the medicine residues again, bring to a boil with strong fire and then decoct with slow fire for 1 h. When the liquid medicine remains about 400 mL, filter to obtain the decoction, and combine the two decoctions to get the finished product.

[0045] Example 4 The preparation method of the traditional Chinese medicine decoction for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury in this example includes the following steps: (1) Put 10 g of Rhizoma Coptidis Rhizoma Zingiberis Recens, 12 g of Pinelliae Rhizoma Praeparatum Cum Zingibere, 20 g of Fructus Trichosanthis Pericarpium, 5 g of Ramulus Cinnamomi, 5 g of Radix Astragali seu Hedysari, and 30 g of Radix Salviae Miltiorrhizae into a medicine pot; (2) Add water according to a water-to-medicine ratio of 9:1, with the liquid level about 5 cm above the medicine surface. Soak for about 35 min, first decoct with strong fire for 50 min, then switch to slow fire and decoct for 1 h. Stir the medicine materials about 3 times during the decocting process to prevent the liquid medicine from overflowing, drying up, or burning. When the liquid medicine remains 1 / 3 of the original volume, filter to obtain the first decoction. Add 800 mL of water to the medicine residues again, bring to a boil with strong fire and then decoct with slow fire for 1 h. When the liquid medicine remains about 400 mL, filter to obtain the decoction, and combine the two decoctions to get the finished product.

[0046] Next, in order to verify the improvement of the traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury in the present invention on coronary microcirculation perfusion and endothelial dysfunction after ischemia-reperfusion injury, the following animal experiments were conducted. I. Experimental animals 40 C57BL / 6 mice (male, 6 - 8 weeks old, body weight 20 - 22 g), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Guangzhou, China, approval number: SCXK(Guangdong)2022 - 0063), were raised in a SPF environment (temperature 22 ± 2°C, humidity 50 ± 10%), and food and water were provided. II. Experimental reagents Nicorandil was purchased from SegMed (SegMed, H20110491, Japan), veterinary isoflurane from Reward (Reward, R510-22-10, China), HE staining solution from Servicebio (China), PBS (pH=7.4) from Sewell (China), ELISA kit for detecting IL-1β, goat anti-rabbit AF488 fluorescent secondary antibody, and goat anti-mouse AF555 fluorescent secondary antibody were all purchased from Invitrogen (Invitrogen, USA), CD31 antibody from Santa (Santa, USA), VE-Cadherin antibody from UpingBio (UpingBio, China), VCAM-1 antibody from Huaan (China), and LC3B antibody from Mitsubishi. III. Experimental Instruments and Consumables Disposable syringe (1 mL) (Kangli Medical Co., Ltd., China), electronic analytical balance (Mettler-Toledo, Switzerland), inverted microscope (Olympus, USA), full-wavelength microplate reader (BioTek, USA), pipette (Eppendorf, Germany), refrigerated high-speed centrifuge (Eppendorf, Germany), fully automated staining and mounting workstation (Leica, Germany), SIMBFI-HR Pro laser speckle analyzer (Simopto, China), small animal super-resolution ultrasound diagnostic instrument (Feinuo, China), laser confocal microscope (ZEISS, Germany), RoVent small animal ventilator (Kent Scientific, USA). IV. Experimental Grouping and Drug Administration Animal grouping and treatment: Mice were divided into 5 groups of 8 each: sham operation group (Sham group), model group (I / R group), low-dose traditional Chinese medicine composition group (JWXXT-L group), high-dose traditional Chinese medicine composition group (JWXXT-H group), and positive control group (Nicorandil group).

[0047] Mice in each group were pre-treated with the drug via gavage for 7 days, once daily. The traditional Chinese medicine (TCM) composition groups (JWXXT-L and JWXXT-H groups) were given the TCM extract powder prepared in Example 2, which was dissolved in physiological saline to form a suspension for gavage administration. The dosages for the low-dose and high-dose TCM composition groups were 13.6 g / kg / d and 27.2 g / kg / d, respectively. Mice in the Sham and model groups were administered physiological saline via gavage at a dose of 0.1 mL / 10 g mouse body weight. The positive control group was administered nicorandil suspension (1.95 mg / kg / d) via gavage.

[0048] The drug administration period for each group of mice was 7 days, and animal modeling was performed 7 days after drug administration. V. Establishing an animal model of myocardial ischemia-reperfusion injury Mice were anesthetized via intraperitoneal injection of 0.5% sodium pentobarbital solution at a rate of 0.1 mL / 10 g body weight. Hair on the chest and armpits was removed with depilatory cream, and the surgical area was disinfected with povidone-iodine. The mice were immobilized and endotracheally intubated to maintain respiration. Successful intubation was indicated by the ventilator frequency matching the chest rise and fall. The mice were placed in a supine position, and the skin on the left chest was cut open, and the muscles were bluntly dissected. The thoracic cavity was opened between the third and fourth ribs to expose the heart. A small portion of the pericardium was torn open below the left atrial appendage to expose the area from the left coronary artery to the left anterior descending artery (LAD). The location of the LAD was then determined under a stereomicroscope. An 8-0 suture was inserted approximately 2 mm below the left atrial appendage, about 0.5 mm deep and 1 mm wide, and ligated to completely block blood flow to the LAD. After ligation, the myocardium below the ligation line turned white, and the electrocardiogram showed persistent ST segment elevation with a convex shape, indicating successful myocardial ischemia. The thoracic cavity was closed with simple sutures. After 45 minutes of ischemia, the cavity was reopened, and the sutures were cut. The surface of the heart changed from pale to red, indicating successful reperfusion. The thoracic cavity was then closed again with sutures. Once the mouse's breathing stabilized, the endotracheal tube was removed, and the mouse was placed on an animal warming blanket until it regained consciousness. During the experiment, the mouse had free access to food and water, its bedding was changed regularly, and humane care was provided.

[0049] Mice in the model group, low-dose group of traditional Chinese medicine composition, high-dose group of traditional Chinese medicine composition, and positive drug group were all modeled according to the above method.

[0050] The procedure for the sham-operated mice was basically the same as before, except that an 8-0 suture was inserted about 2 mm below the left atrial appendage, about 0.5 mm deep and about 1 mm wide, and then immediately pulled out. The left atrial appendage (LAD) was not ligated. The thoracic cavity was then sutured closed. After the mice’s breathing stabilized, the endotracheal tube was removed and the mice were placed on an animal warming blanket until they woke up.

[0051] VI. Statistical Methods SPSS 27.0 statistical software was used. Quantitative data results are expressed as mean ± standard deviation (SD). The mean ± standard deviation (±s) is used to represent the median (first quartile to third quartile) [M (P25 to P75)]. For normal distributions, one-way ANOVA is used for comparisons between groups. Homogeneity of variance is determined by the Levene test. When variances are homogeneous, the Bonferroni test is used for multiple comparisons; when variances are unequal, the Dunnett's T3 test is used. For non-normal distributions, nonparametric tests are used, and the Kruskal-Wallis H test is used for multiple comparisons. P <0.05 indicates a statistically significant difference. VII. Determination of coronary microangiopathy phenotype caused by ischemia-reperfusion injury Effusion (EF) and blood flow (FS) values ​​in mice were observed using ultrasound. Myocardial perfusion blood flow was detected using laser speckle imaging. Mouse heart tissue was collected, stored at -80℃ or embedded, and subjected to HE staining and immunofluorescence staining for observation. Mouse serum was collected, stored at -80℃, and IL-1β levels were detected using ELISA.

[0052] 1. Echocardiography to assess cardiac function Mice were anesthetized with 2% isoflurane / oxygen inhalation. The chest was shaved to fully expose the skin, and the mice were fixed to a plate at a constant temperature of 37°C. An ultrasound coupling agent was applied to the ultrasound probe, and a research-grade D6 LAB color Doppler ultrasound diagnostic instrument (probe frequency 30MHz, detection depth 10-15mm) was used for detection. The probe was placed in the left chest of the mouse, and B-mode ultrasound was used to record the long and short axis views of the left ventricle. Then, M-mode ultrasound was used to record the motion of the left ventricle at the papillary muscle level. Left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) were measured. Ten cardiac cycles were measured for each mouse, and the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated.

[0053] 2. Laser speckle imaging for detecting cardiac perfusion blood flow The cardiac blood flow of mice was imaged using a laser speckle analysis system (SIMBFI-HR Pro, Simopto, China). The specific steps are as follows: (1) Anesthetize mice with 2% isoflurane / oxygen inhalation and maintain the body temperature of the mice at 36.5±0.5℃ using a heating blanket. Fix the mice in a supine position on a flat black background board, cut open the thoracic cavity to fully expose the heart, and gently clean the surface of the heart with sterile saline.

[0054] (2) Blood perfusion images were acquired using a SIMBFI-HR laser speckle analysis system (including a charge-coupled device). The camera was positioned 15 cm above the mouse heart, and the focus was adjusted to achieve optimal image clarity. Raw speckle images of the heart were captured at a resolution of 100 consecutive images per second, a sampling frequency of 35 Hz, and a sampling time of 30 seconds. False-color images of perfusion were obtained, with blue to red representing cardiac vascular perfusion from low to high. The perfusion blood flow in the region of interest (ROI) at the apex of the left ventricle of the mouse was analyzed, and the average perfusion volume was calculated.

[0055] 3. HE staining HE staining was used to examine the pathological condition of mouse myocardial tissue. The specific experimental steps are as follows: (1) Take frozen sections of mouse heart.

[0056] (2) Hematoxylin staining of cell nuclei: Slices are stained with Harris hematoxylin staining solution for about 5 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

[0057] (3) Eosin staining of cytoplasm: Slices are stained in eosin staining solution for 1-3 min.

[0058] (4) Dehydration and mounting: Place the sections in 95% alcohol I for 5 min - 95% alcohol II for 5 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - xylene I for 5 min - xylene II for 5 min to dehydrate and become transparent. Remove the sections from the xylene and let them dry slightly. Then mount them with neutral resin.

[0059] (5) Microscopic examination, image acquisition and analysis.

[0060] 4. Detection of serum IL-1β levels using ELISA method Add 100 µL of serum to each reaction well and incubate at 37°C for 1-2 hours. After discarding the liquid and washing, add 100 µL of antibody working solution to each well and incubate at 37°C for 1 hour. Wash three times. Then add 100 µL of enzyme conjugate working solution and incubate at 37°C in the dark for 30 minutes. Wash three times. Finally, add 100 µL of TMB substrate solution and react at 37°C in the dark for 10-30 minutes, until a clear color gradient appears. Terminate the reaction. Within 10 minutes, use a microplate reader with a detection wavelength of 450 nm to measure the OD value of each well and calculate the IL-1β concentration of each well.

[0061] 5. Immunofluorescence staining The expression levels of VE-Cadherin, VCAM-1, and LC3B in cardiac vascular endothelial cells were observed using the following procedures: (1) Take frozen sections of mouse heart with a thickness of 6 μm.

[0062] (2) Tissue fixation: Pre-cool acetone, immerse the heart sections completely in ice acetone, fix in a -20℃ refrigerator for 15 min, then wash with cold PBS 3 times, 5 min each time, and place the sections in a humidified box after washing.

[0063] (3) Permeation: Wipe the moisture around the tissue dry, circle the tissue area with an immunohistochemical pen, then add 0.2% Triton solution and permeate at room temperature for 10 min, then wash with PBS 3 times, 5 min each time.

[0064] (4) Blocking: Remove the water from the slide, add 10% goat serum to the tissue to completely cover it, and block at room temperature for 1 hour. After blocking, remove the blocking solution.

[0065] (5) Incubation of primary antibody: Add the prepared primary antibody solutions, CD31 (dilution ratio 1:300), VE-Cadherin (dilution ratio 1:300), and VCAM-1 (dilution ratio 1:50) to the tissue respectively, and incubate at 4℃ for 14-16h. Then, recover the primary antibody and wash with PBS 3 times for 5min each time.

[0066] (6) Incubation of secondary antibody: Remove the water from the slide and, in the dark, drop the diluted secondary antibody solution, including AF488 fluorescent secondary antibody (dilution ratio 1:500) and AF555 fluorescent secondary antibody (dilution ratio 1:500), onto the tissue to completely cover it. Incubate at room temperature in the dark for 1 hour, then remove the secondary antibody and wash with PBS 3 times for 5 minutes each time.

[0067] (7) Mounting: Carefully wipe away the immunohistochemistry pen marks with anhydrous ethanol, avoiding contact with the tissue. After wiping away the immunohistochemistry ring, shake off the moisture from the slide. Add a DAPI-containing anti-fluorescence quenching mounting medium to the tissue, avoiding air bubbles. Carefully cover with a coverslip and mount with nail polish.

[0068] (8) Use laser confocal microscope to take pictures and use ImageJ software to analyze the fluorescence colocalization and fluorescence intensity in vascular endothelium.

[0069] VIII. Experimental Results 1. Effects of a traditional Chinese medicine composition on cardiac function in mice with myocardial ischemia-reperfusion injury M-mode ultrasound images and cardiac function of mice in each group are as follows: Figure 1 As shown in the figure, A is an M-mode echocardiogram of the left ventricle in mice; B is a statistical graph of LVEF in mice; and C is a statistical graph of LVFS in mice. Compared with the Sham group, ### P <0.001, #### P <0.0001; compared with the I / R group, ** P <0.01, *** P <0.001.

[0070] from Figure 1 As can be seen from the ultrasound results in mice, compared with the sham-operated group, the LVEF and LVFS of the heart in the model group mice were significantly decreased. Compared with the model group, the LVEF and LVFS of the heart in the mice in the traditional Chinese medicine composition groups (i.e., the JWXXT-L group and the JWXXT-H group) were significantly increased, indicating that the traditional Chinese medicine composition of the present invention has a protective effect on myocardial contractile function in mice in myocardial ischemia-reperfusion injury.

[0071] 2. Effects of Traditional Chinese Medicine Combinations on Pathological Changes in Myocardial Tissue of Mice with Myocardial Ischemia-Reperfusion Injury HE staining of heart tissue from each group of mice is as follows: Figure 2 As shown.

[0072] from Figure 2 As can be seen from the HE staining results, the model group mice showed significant myocardial infarction damage, exhibiting pathological features such as disordered myocardial fiber arrangement, myocardial swelling, vascular endothelial edema, luminal narrowing, and increased peripheral inflammatory infiltration. In contrast, the myocardial fiber disorder in the traditional Chinese medicine composition groups (i.e., JWXXT-L and JWXXT-H groups) and the Nicorandil group mice was improved, with reduced myocardial and perivascular inflammatory infiltration and luminal narrowing. This indicates that the traditional Chinese medicine composition of this invention has a protective effect on the myocardium and blood vessels in myocardial ischemia-reperfusion injury, and can alleviate pathological conditions such as myocardial and perivascular inflammatory infiltration.

[0073] 3. Effects of traditional Chinese medicine composition on cardiac blood flow perfusion in mice with myocardial ischemia-reperfusion injury Cardiac blood perfusion status of mice in each group as follows Figure 3 As shown in the figure, A represents the cardiac perfusion map of each group of mice; B represents the statistical graph of the average cardiac blood flow of each group of mice. Compared with the Sham group, ## P <0.01; compared with the I / R group, * P <0.05.

[0074] from Figure 3 As can be seen from the laser speckle results, the mean blood perfusion of the heart vessels in the model group mice was significantly reduced compared with the sham-operated group. Compared with the model group, the mean blood perfusion of the heart vessels in the mice in the traditional Chinese medicine composition groups (i.e., JWXXT-L group and JWXXT-H group) and Nicorandil group was significantly increased, suggesting that the traditional Chinese medicine composition of the present invention has a protective effect on the heart in myocardial ischemia-reperfusion injury and can increase the blood perfusion of the heart vessels in mice with myocardial ischemia-reperfusion injury.

[0075] 4. Effects of traditional Chinese medicine composition on VCAM-1, an adhesion molecule on the surface of endothelial cells in mice with myocardial ischemia-reperfusion injury. VCAM-1 expression in cardiac endothelial cells of mice in each group is as follows: Figure 4 As shown.

[0076] from Figure 4As can be seen from the immunofluorescence assay, compared with the Sham group, the expression level of VCAM-1, an adhesion molecule on the surface of vascular endothelial cells, was increased in the I / R group mice due to myocardial ischemia-reperfusion injury. However, compared with the I / R group, the expression of VCAM-1 in the cardiac vascular endothelium of the herbal composition groups (i.e., JWXXT-L and JWXXT-H groups) and the positive control group was decreased, suggesting that the herbal composition of this invention has a protective effect on the heart and microvessels in myocardial ischemia-reperfusion injury, possibly related to its reduction of the expression of VCAM-1, a biomarker of inflammation and vascular lesions, thereby helping to alleviate the adhesion and infiltration of inflammatory cells to the vascular endothelium.

[0077] 5. Effects of a traditional Chinese medicine composition on VE-Cadherin, a cell-connecting protein in mice with myocardial ischemia-reperfusion injury. VE-Cadherin is a linker protein representing the barrier function of microvessels, helping to reduce the migration of inflammatory cells across blood vessels and vascular pathological changes. The expression levels of VE-Cadherin in the cardiac endothelial cells of different groups of mice are shown below. Figure 5 As shown.

[0078] from Figure 5 As can be seen from the immunofluorescence assay, compared with the Sham group, the expression of VE-Cadherin in vascular endothelial cells was weakened in the I / R group mice due to myocardial ischemia-reperfusion injury. However, compared with the I / R group, the expression of VE-Cadherin in vascular endothelial cells of the traditional Chinese medicine composition groups (i.e., JWXXT-L group and JWXXT-H group) and the positive control group was enhanced, suggesting that the traditional Chinese medicine composition of this invention has a protective effect on the heart and microvessels in myocardial ischemia-reperfusion injury, which may be related to its increase in the intercellular junction protein VE-Cadherin, thereby helping to maintain microvascular barrier function and reduce structural and functional damage to the vascular endothelium.

[0079] 6. Effects of traditional Chinese medicine composition on LC3B expression in endothelial cells of mice with myocardial ischemia-reperfusion injury The expression levels of LC3B in the cardiac vascular endothelium of mice in each group are as follows: Figure 6 As shown in the figure, A is the immunofluorescence map of cardiac tissue; B is the statistical graph of relative fluorescence intensity of LC3B; and C is the statistical graph of colocalization coefficients of CD31-LC3B. Compared with the Sham group, # P <0.05; compared with the I / R group, * P <0.05, ** P <0.01.

[0080] from Figure 6As can be seen from the immunofluorescence assay, compared with the Sham group, the expression of LC3B in vascular endothelial cells was weakened in the I / R group mice due to myocardial ischemia-reperfusion injury. However, compared with the I / R group, the expression of LC3B in vascular endothelial cells of the traditional Chinese medicine composition groups (i.e., JWXXT-L group and JWXXT-H group) and the positive control group was enhanced, suggesting that the traditional Chinese medicine composition of this invention has a protective effect on the heart and microvessels in myocardial ischemia-reperfusion injury, which may be related to its improvement of LC3B levels in endothelial cells.

[0081] In summary, the traditional Chinese medicine composition of the present invention can protect the integrity of vascular endothelial structure and barrier function, reduce myocardial inflammatory infiltration, improve cardiac function, and restore normal vascular blood flow perfusion, thereby preventing and treating coronary microangiopathy caused by ischemia-reperfusion.

[0082] Then, in order to verify the regulation of endothelial cell mitochondrial autophagy by the traditional Chinese medicine composition of the present invention for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury, the following cell experiments were also conducted.

[0083] I. Experimental Reagents High-glucose DMEM medium (containing double antibodies) was purchased from KGI Biotech (KGI Biotech, China); glucose-free DMEM medium was purchased from Procell (Procell, China); fetal bovine serum and trypsin were purchased from Gibco (Gibco, USA); the Tunel apoptosis detection kit, mitochondrial red fluorescent probe, lysosomal green fluorescent probe, and anti-fluorescence quenching mounting medium were purchased from Beyotime (Beyotime, China); the ROS fluorescent probe was purchased from Meilunbio (Meilunbio, China); goat serum for blocking was purchased from Solarbio (Solarbio, China); goat anti-rabbit AF488 fluorescent secondary antibody and goat anti-mouse AF555 fluorescent secondary antibody were purchased from Invitrogen (Invitrogen, USA); and CD31 antibody was purchased from Santa (Santa, USA). VCAM-1 antibody and VE-Cadherin antibody were purchased from Huaan (China), LC3B antibody was purchased from Proteintech (USA), PINK1 antibody and Parkin antibody were purchased from Abmart (China), and β-Actin antibody, P62 antibody and 10×RIPA were purchased from CST (USA).

[0084] II. Grouping, Modeling, and Drug Administration Human coronary artery endothelial cells (HCAECs) were divided into 5 groups: blank control group (Control group), oxygen-glucose deprivation and reoxygenation model group (OGD / R group), JWXXT-L group, JWXXT-M group, and JWXXT-H group.

[0085] Drug intervention and OGD / R modeling: The cell culture medium of the JWXXT-L group, JWXXT-M group, and JWXXT-H group was replaced with high-glucose DMEM medium containing the herbal extract powder from Example 2 at the corresponding concentrations (drug concentration of 20 µg / mL for JWXXT-L group, 40 µg / mL for JWXXT-M group, and 80 µg / mL for JWXXT-H group). The Control group and OGD / R group were only replaced with high-glucose DMEM medium. After culturing in a cell culture incubator at 37°C and 5% CO2 for 16 h, the OGD / R group, JWXXT-L group, JWXXT-M group, and JWXXT-H group were all replaced with sugar-free DMEM medium and placed in a hypoxic incubator (37°C, 94% H2, 5% CO2, 1%). After 4 hours of oxygen and glucose deprivation (O2), the medium was replaced with normal high-glucose DMEM medium or high-glucose DMEM medium containing the corresponding concentration of the Chinese herbal extract powder from Example 2. The medium was then reoxygenated in a cell culture incubator (37°C, 5% CO2) for 1 hour to simulate in vitro coronary microvascular endothelial cell injury induced by myocardial ischemia-reperfusion injury.

[0086] III. Experimental Methods 1. Detect cell viability and fluorescent TUNEL staining in each group. (1) Cell viability was detected by CCK8 assay. The specific steps are as follows: Human coronary endothelial cells (HCAECs) were used at a concentration of 5 × 10⁻⁶. 3 Cells were seeded at a density of 100 cells / mL in 96-well plates. After drug intervention and OGD / R modeling in each group, the cell culture medium was discarded, and 100µL of fresh high-glucose DMEM medium and 10µL of CCK8 reagent were added to each well. The mixture was stirred and incubated in the dark for 1-4 hours. The detection wavelength was set to 450nm, and the OD values ​​of each group were detected in a microplate reader.

[0087] (2) Fluorescent TUNEL staining was used to detect apoptosis in each group. The specific steps for detection are as follows: Cell fixation: Remove the culture medium, wash the cell slides once with pre-cooled PBS, fix with 4% paraformaldehyde at room temperature for 30 minutes, and then wash once with PBS.

[0088] Permeability test: Incubate with PBS containing 0.3% Triton X-100 at room temperature for 5 minutes, then wash twice with PBS.

[0089] Staining: Cut circular slices of the same size as 24-well plates. Add 50 µL of Tunel detection solution to each cell smear and cover the smear with the circular slices to ensure that the reaction solution fully covers the cell surface. Incubate at 37°C in the dark for 60 min. Then wash three times with PBS for 5 min each time to remove unbound dUTP.

[0090] Mounting: Mount the slides with a DAPI-containing anti-fluorescence quenching mounting medium.

[0091] Image capture: Observation was performed under a fluorescence microscope with the excitation wavelength range of 450-500 nm and the emission wavelength range of 515-565 nm. Fluorescence images were captured. Apoptotic cell nuclei showed obvious green fluorescence, while normal cells showed no signal. ImageJ software was used to analyze the cell fluorescence intensity.

[0092] 2. Detect changes in ROS levels and mitochondrial membrane potential in each group of cells. (1) DCFH-DA method for detecting cell ROS level: After drug intervention and OGD / R modeling treatment, the cell culture medium was removed, and the prepared DCFH-DA solution was added to cover the cells. The cells were incubated in a 37℃ incubator in the dark for 20-30 min. The cells were then washed three times with serum-free cell culture medium for 1 min each time to fully remove the DCFH-DA that had not entered the cells. Finally, serum-free culture medium was added, and the OD value of each group was detected under a fluorescence microplate reader with an excitation wavelength of 504 nm and an emission wavelength of 529 nm.

[0093] (2) The fluorescent probe method was used to detect the mitochondrial membrane potential level. The specific detection steps are as follows: After drug intervention and OGD / R modeling, the culture medium was aspirated, the cells were washed once with PBS, and 1 mL of cell culture medium containing JC-1 working solution was added (cell culture medium: JC-1 staining working solution = 1:1, mixed thoroughly). The cells were incubated at 37°C in the dark for 20 minutes. After incubation, the supernatant was aspirated, and the cells were gently washed twice with JC-1 staining buffer (1×) preheated to 37°C to remove unbound JC-1. Finally, 1 mL of cell culture medium was added, and the cells were detected under a laser confocal microscope. The intensity of red and green fluorescence was analyzed, and the red / green fluorescence ratio was calculated to assess changes in mitochondrial membrane potential.

[0094] 3. Fluorescence co-localization detection of mitochondrial phagosome levels in each group of cells After drug intervention and OGD / R modeling, the cell culture medium was removed, and the prepared staining working solution (50 nM Lyso Tracker Green + 50 nM Mito Tracker Red solution) was added. The cells were co-incubated at 37°C for 15-20 min, then the staining working solution was removed, and the cells were washed twice with fresh serum-free culture medium. Images were taken using a laser confocal microscope, and the fluorescence intensity was analyzed using ImageJ software.

[0095] 4. Western blot analysis was performed to detect the protein expression levels in each group of cells. (1) Lysis of cells for protein extraction: All operations were performed on ice. After removing the cell culture medium, the cells were washed twice with ice-cold PBS, the PBS was thoroughly aspirated, cell lysis buffer was added, and the cells were lysed on ice for 30 min. The cells were scraped and transferred to EP tubes, and the cell suspension was centrifuged at 12,000 rpm for 15 min at 4 °C. After centrifugation, the supernatant was transferred to a new EP tube.

[0096] (2) BCA method for protein concentration detection: Take a 96-well plate and add 20 µL of standard protein solution (concentrations of 0, 25, 50, 100, 200, 300, 400, and 500 µg / mL) in sequence. Add 2 µL of the protein sample to be detected in the other wells in sequence, and add ultrapure water to make up to 20 µL. Set up two replicates for each sample and standard curve. Then add 200 µL of BCA working solution to all wells, and incubate in a 37°C incubator for 30 min. Detect the absorbance of all wells at a wavelength of 562 nm using a full-wavelength microplate reader, calculate the standard curve, and calculate the concentration of each sample based on the standard curve.

[0097] (3) Protein denaturation: After adding loading buffer (5X) to each sample, place it in a 98℃ metal bath for 5 min to denature the protein.

[0098] (4) Electrophoresis: Prepare SDS polyacrylamide gel, load the sample into the well of the gel, and ensure that the sample volume and total protein amount are consistent for all samples. Set the voltage to 80V and let the protein electrophore in the stacking gel until the molecular weight standard marker color begins to separate. Then adjust the voltage to 100V and observe that the bromophenol blue dye in the sample reaches the bottom of the gel, and then stop electrophoresis.

[0099] (5) Electroporation: Activate the PVDF membrane by soaking it in methanol beforehand. Prepare the transfer sponge, transfer filter paper, and transfer clamp to form a transfer "sandwich" structure. Pour the transfer solution into a dish and wet the transfer "sandwich". Remove the electrophoresis gel, cut off the stacking gel, carefully remove the separating gel and place it on the transfer filter paper. Cover the activated PVDF membrane on the separating gel, avoiding air bubbles. Secure the transfer clamp in the transfer electrode holder, with the positive electrode facing the positive electrode and the negative electrode facing the negative electrode. Place the transfer holder into the electrophoresis tank, add the electroporation solution, place the electrophoresis tank in an ice box, and cover it with ice for low-temperature transfer. The transfer conditions are constant current transfer with a current of 300mA. Set the transfer time according to the molecular weight of the target protein.

[0100] (6) Blocking: After the transfer is completed, remove the PVDF membrane, discard the gel, and completely soak the PVDF membrane in TBST solution containing 5% skim milk powder. Place it on a shaker at room temperature and slowly block it for 1.5 hours to block the positions on the membrane where no protein has been adsorbed.

[0101] (7) Membrane cutting: After the blocking is completed, remove the blocking solution, wash the membrane three times with TBST buffer on a shaker, and then cut the membrane band containing the target protein according to the marker position.

[0102] (8) Primary antibody incubation: Dilute the primary antibody with diluent to the following working concentrations: anti-LC3 (1:1000), anti-Beclin1 (1:1000), anti-PINK1 (1:1000), anti-Parkin (1:1000), anti-VCAM-1 (1:1000), anti-VE-Cadherin (1:1000), anti-β-actin (1:1000), and anti-GAPDH (1:1000). Incubate the corresponding target protein bands with the corresponding primary antibody dilution solution overnight at 4°C with gentle shaking to allow the primary antibody to better collide and bind with the target protein. The next day, recover the primary antibody and wash the membrane three times with TBST for 6 minutes each time.

[0103] (9) Secondary antibody incubation: Incubate the band with secondary antibody (1:3000) at room temperature for 1-2 hours, then wash the membrane with TBST 3 times, 6 min each time.

[0104] (10) Development: Visual detection was performed using an enhanced chemiluminescence detection kit. In a darkroom, developer solutions A and B (luminol and hydrogen peroxide, respectively) were mixed in a 1:1 ratio. The bands were immersed in the developer solution, and the bands were developed and photographed using a gel imaging system. The gray values ​​of the target protein in the images were quantified using Image-Pro Plus 6.0 software and normalized with the gray values ​​of the internal control. All experiments were repeated three times.

[0105] IV. Statistical Methods SPSS 27.0 statistical software was used. Quantitative data results are expressed as mean ± standard deviation (SD). The mean ± standard deviation (±s) is used to represent the median (first quartile to third quartile) [M (P25 to P75)]. For normal distributions, one-way ANOVA is used for comparisons between groups. Homogeneity of variance is determined by the Levene test. When variances are homogeneous, the Bonferroni test is used for multiple comparisons; when variances are unequal, the Dunnett's T3 test is used. For non-normal distributions, nonparametric tests are used, and the Kruskal-Wallis H test is used for multiple comparisons. P <0.05 indicates a statistically significant difference. V. Experimental Results 1. Effects of traditional Chinese medicine composition on endothelial cell viability and apoptosis The results of cell viability and apoptosis of endothelial cells in each group are as follows: Figure 7 As shown in the figure, A represents the morphological changes of cells in each group observed under a light microscope; B represents the cell viability level of cells treated with different drug concentrations without OGD / R modeling, as detected by CCK8; C represents the cell viability level of cells treated with different drug concentrations after OGD / R modeling, as detected by CCK8; D represents the TUNEL fluorescence spectrum of cells in each group; and E represents the TUNEL fluorescence statistical graph of cells in each group. Compared with the Control group, ## P <0.01, ### P <0.001; compared with the OGD / R group, * P <0.05, ** P <0.01, *** P <0.001.

[0106] from Figure 7 It can be seen that, compared with the blank control group, the cell viability of the model group was significantly reduced, the cell morphology was significantly changed, and the cell apoptosis rate was increased. However, after intervention with the traditional Chinese medicine composition of the present invention, the cell viability was significantly improved, the normal morphology of the cells was restored, and the cell apoptosis rate was reduced at a concentration of 5-160 µg / mL, indicating that the traditional Chinese medicine composition of the present invention can significantly protect the activity of endothelial cells.

[0107] 2. Effects of Traditional Chinese Medicine Combinations on Endothelial Cell Function The expression levels of VE-Cadherin and VCAM-1 proteins in cells of each group are as follows: Figure 8 As shown in the figure, compared with the Control group, # P <0.05; compared with the OGD / R group, *P <0.05, ** P <0.01.

[0108] from Figure 8 As can be seen, compared with the blank control group, the expression level of VE-Cadherin in the model group cells was significantly reduced, while the expression of VCAM-1 was significantly increased. After intervention with the traditional Chinese medicine composition of the present invention, the expression level of VE-Cadherin in endothelial cells was significantly increased, and the expression level of VCAM-1 protein was significantly decreased, suggesting that the traditional Chinese medicine composition of the present invention can protect the endothelial cell barrier function and reduce the expression of inflammation-related adhesion molecules.

[0109] 3. Effects of traditional Chinese medicine composition on ROS and mitochondrial membrane potential of endothelial cells Excessive ROS can lead to mitochondrial dysfunction, resulting in a decrease in mitochondrial membrane potential. The extent of mitochondrial damage in each group of cells is as follows: Figure 9 As shown in the figure, A is the fluorescence image of mitochondrial membrane potential in each group; B is the statistical graph of mitochondrial membrane potential in each group; C is the ROS level of cells in each group. Compared with the control group, ## P <0.01, ### P <0.001; compared with the OGD / R group, * P <0.05, ** P <0.01.

[0110] from Figure 9 As can be seen, compared with the blank control group, the ROS level in the model group cells was significantly increased, and the mitochondrial membrane potential was significantly decreased. However, after intervention with the traditional Chinese medicine composition of this invention, the ROS level in cells significantly decreased, and the mitochondrial membrane potential significantly increased. This indicates that the traditional Chinese medicine composition of this invention can reduce the expression of ROS in endothelial cells and increase the mitochondrial membrane potential, thereby reducing mitochondrial damage.

[0111] 4. Effects of traditional Chinese medicine composition on mitochondrial lysosomes in different groups of cells Co-localization of mitochondria and lysosomes in cells of each group is as follows: Figure 10 As shown in the figure, A represents fluorescence images of mitochondria and lysosomes; B represents a qualitative descriptive diagram of mitochondria and lysosomes; and C represents a statistical diagram of co-localization of fluorescence in mitochondria and lysosomes. Compared with the Control group, ### P <0.001; compared with the OGD / R group, *** P <0.001.

[0112] from Figure 10It can be seen that, compared with the blank control group, the number of lysosomes in the model group cells was significantly reduced, and the fluorescence co-localization with mitochondria was significantly reduced. However, after intervention with the traditional Chinese medicine composition of this invention, the number of lysosomes significantly increased, and the co-localization of lysosomes with mitochondria significantly increased. This indicates that the traditional Chinese medicine composition of this invention may increase the number of mitophagic lysosomes, enabling the degradation of mitochondria damaged by oxygen-glucose deprivation and reoxygenation induced by lysosomes.

[0113] 5. Effects of traditional Chinese medicine compositions on mitochondrial autophagy-related proteins in different cell groups The expression of mitochondrial autophagy-related proteins in cells of each group are as follows: Figure 11 As shown in the figure, compared with the Control group, # P <0.05, ## P <0.01; compared with the OGD / R group, * P <0.05, ** P <0.01, *** P <0.001.

[0114] from Figure 11 As can be seen, compared with the blank control group, the LC3-II / LC3-I ratio, PINK1 and Parkin protein expression were significantly decreased, while the P62 protein expression level was significantly increased in the model group cells. However, after intervention with the traditional Chinese medicine composition of this invention, the LC3-II / LC3-I ratio, PINK1 and Parkin protein expression were significantly increased, while the P62 protein expression level was significantly decreased. This suggests that the traditional Chinese medicine composition of this invention may enhance the level of mitochondrial autophagy in endothelial cells after oxygen-glucose deprivation-reoxygenation injury by activating the PINK1 / Parkin signaling pathway.

[0115] In summary, the traditional Chinese medicine composition of the present invention may improve endothelial dysfunction, maintain vascular endothelial barrier function, further reduce vascular inflammation and endothelial hyperpermeability by regulating mitochondrial autophagy, thereby reducing mitochondrial damage and oxidative stress in endothelial cells, and ultimately improve cardiac microvascular blood perfusion and cardiac function, thus playing a role in preventing and treating coronary microangiopathy caused by ischemia-reperfusion injury.

[0116] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury, characterized in that, The raw materials, by weight, include: 10-20 parts of Coptis chinensis, 9-15 parts of Pinellia ternata, 15-30 parts of Trichosanthes kirilowii peel, 5-15 parts of Cinnamomum cassia, 5-30 parts of Astragalus membranaceus, and 15-30 parts of Salvia miltiorrhiza.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The raw materials, by weight, include: 15 parts of Coptis chinensis, 15 parts of Pinellia ternata, 30 parts of Trichosanthes kirilowii peel, 15 parts of Cinnamomum cassia, 30 parts of Astragalus membranaceus, and 15 parts of Salvia miltiorrhiza.

3. The traditional Chinese medicine composition according to claim 1 or 2, characterized in that, Pharmaceutically acceptable excipients can also be added.

4. The traditional Chinese medicine composition according to claim 1 or 2, characterized in that, The dosage form of the traditional Chinese medicine composition is decoction, oral solution, powder of traditional Chinese medicine extract, powder, granule, tablet, pill or capsule.

5. A method for preparing the traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury as described in any one of claims 1-4, characterized in that, Includes the following steps: Add water according to the water-to-medicine ratio of (8-10):1, soak for 30-40 minutes, first decoct over high heat for 30-60 minutes, then simmer over low heat for 1-1.5 hours, stirring the herbs 2-3 times during the decoction process, filter the first decoction, add 8-10 times the weight of water to the dregs again, bring to a boil, then simmer over low heat for 1-1.5 hours, filter the decoction, and combine the two filtrates to obtain the final product.

6. The use of the traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury as described in any one of claims 1-4 in the preparation of a medicament for preventing and treating coronary microvascular disease.

7. The application according to claim 6, characterized in that, The coronary microangiopathy mentioned above is coronary microangiopathy caused by ischemia-reperfusion injury.

8. The use of the traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury as described in any one of claims 1-4 in the preparation of a drug for preventing and treating structural and functional disorders of coronary microvascular endothelium.

9. The use of the traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury as described in any one of claims 1-4 in the preparation of a drug for preventing and treating coronary slow flow syndrome.

10. The use of the traditional Chinese medicine composition for preventing and treating coronary microvascular disease caused by ischemia-reperfusion injury as described in any one of claims 1-4 in the preparation of a drug for preventing and treating myocardial infarction and microvascular dysfunction heart failure.

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