Application of ligustilide in preparation of medicine for relieving inflammatory response after myocardial ischemia

Abstract

The invention belongs to the technical field of cardiovascular disease treatment medicines, and particularly relates to application of ligustilide in preparation of a medicine for relieving inflammatory response after myocardial ischemia. According to the application disclosed by the invention, research finds that ligustilide can be used for remarkably inhibiting the level of S100A9 in serum, remarkably inhibiting expression of S100A9 mRNA and protein in a cardiac infarction region and expression of S100A9 in neutrophil for the first time, so that release of inflammatory factors and chemotactic factors can be effectively reduced, and occurrence of myocardial tissue inflammatory response is prevented or inflammatory response caused by myocardial ischemia is relieved; the compound can inhibit MI mouse infarction region neutrophil and mononuclear cell infiltration, and inhibit formation of NETs after MI and expression and activation of NLRP3 inflammasome, so that inflammatory response after MI can be relieved, and myocardial tissue can be protected. The application range of ligustilide in cardiovascular diseases is expanded, and a new drug choice is provided for prevention and treatment of inflammatory response after myocardial ischemia.

Description

Technical Field

[0001] This invention belongs to the field of cardiovascular disease treatment drug technology, specifically relating to the application of ligustilide in the preparation of drugs that reduce inflammatory response after myocardial ischemia. Background Technology

[0002] Myocardial infarction (MI) has seen a rapid increase in incidence and mortality over the past few decades, becoming one of the most significant threats to human health. Following MI, cardiomyocytes undergo various pathological changes, including metabolic disturbances, impaired calcium homeostasis, excessive secretion of reactive oxygen species, inflammatory responses, myocardial fibrosis, ventricular remodeling, and cardiomyocyte apoptosis, leading to myocardial ischemia and hypoxia, and a significant decline in cardiac function. Although reperfusion therapy strategies such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) have improved the survival rate of MI patients, many still suffer from severe cardiac remodeling and heart failure, impacting their prognosis.

[0003] Myocardial ischemia triggers pathological inflammation in the heart. The levels of inflammatory factors are directly related to the damage to cardiac function and the number of dead cells after ischemia. A persistently dysregulated inflammatory response can exacerbate myocardial tissue damage, leading to myocardial ischemia and eventually heart failure. S100A8 / A9 accounts for approximately 45% of all cytoplasmic proteins in neutrophils. In the acute phase of myocardial infarction (MI), S100A8 / A9 is rapidly released after ischemic injury. Its levels rise in the coronary arteries and systemic circulation before the appearance of markers of myocardial injury such as myoglobin and troponin. S100A8 / A9 is an endogenous activator of receptors for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4), which can lead to the activation of endothelial and immune cells, promote the massive release of various inflammatory factors (such as IL-1β and TNF-α) and chemokines, attract more immune cells (such as neutrophils and macrophages) to the infarcted area, and exacerbate the local inflammatory response, leading to further damage to cardiomyocytes and inducing cardiomyocyte apoptosis. Studies have shown that elevated serum S100A8 / A9 levels are significantly associated with an increased risk of heart failure after acute myocardial infarction (MI) and are an important driving factor in the development and progression of heart failure.

[0004] Since the inflammatory response is involved in the pathological process of myocardial ischemia (MI), regulating the inflammatory response is an effective means of intervention. Although there are many commercially available anti-inflammatory drugs, the pathological inflammation in this heart disease is an aseptic inflammatory response. Although it shows similarities with inflammation caused by exogenous infection in some test indicators, the triggering mechanism is different. Therefore, the treatment drugs are not the same, and more available drugs need to be developed for the aseptic inflammatory response caused by myocardial ischemia. Summary of the Invention

[0005] To address the above-mentioned technical problems, this invention provides the application of ligustilide in the preparation of drugs that alleviate inflammatory responses after myocardial ischemia. This invention is the first to discover that ligustilide can reduce S100A9 mRNA and protein expression in infarcted myocardial tissue and serum S100A9 levels in mice with myocardial infarction. Furthermore, this invention finds that ligustilide can inhibit neutrophil and monocyte infiltration in the infarcted area of ​​MI mice, as well as NETs formation and NLRP3 inflammasome expression and activation after MI, demonstrating its potential to prevent or alleviate inflammatory responses after myocardial ischemia.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides the use of ligustilide in the preparation of medicaments for preventing or reducing inflammatory responses following myocardial ischemia, wherein the functional components of the medicaments include ligustilide.

[0007] Ligusticolone (Lig) is a natural active ingredient. This invention, through a mouse model of myocardial ischemia, found that ligusticolone can significantly inhibit serum S100A9 levels, significantly inhibit S100A9 mRNA and protein expression in the infarcted area of ​​the heart, and S100A9 expression in neutrophils. This effectively reduces the release of inflammatory factors and chemokines, preventing or alleviating inflammatory responses in myocardial tissue caused by myocardial ischemia. Furthermore, the results demonstrate that ligusticolone can significantly reduce the infiltration of neutrophils and monocytes, significantly inhibit the formation of extracellular neutrophil traps (NETs) and the assembly of NLRP3 inflammasomes, reduce Caspase-1 activation, and decrease the mRNA expression of IL-1β and TNF-α, thereby alleviating the inflammatory response after myocardial ischemia and protecting myocardial tissue.

[0008] Furthermore, this invention also demonstrates that ligustilide has a dose-gradient effect in reducing the infarct area of ​​the heart in MI mice within a certain dose range, and can improve the diastolic and systolic function of the ventricles, thereby improving cardiac function. It can also downregulate the serum levels of CK, CK-MB, and LDH in the early stage of myocardial ischemia, and significantly reduce the number of apoptotic cardiomyocytes caused during MI injury, demonstrating that ligustilide has a certain therapeutic effect on myocardial injury caused by MI.

[0009] Preferably, the myocardial ischemia is acute myocardial ischemia.

[0010] Preferably, the drug is a drug for preventing inflammatory response after myocardial ischemia.

[0011] Preferably, the drug is a drug that inhibits the level of S100A9 in serum.

[0012] Preferably, the drug is a drug that inhibits the expression of S100A9 mRNA and protein in the myocardial infarction region.

[0013] Preferably, the drug is a drug that inhibits the expression of S100A9 in neutrophils.

[0014] Preferably, the dosage form of the drug is an oral preparation.

[0015] More preferably, the drug further contains pharmaceutically acceptable excipients.

[0016] The beneficial effects of this invention are as follows: This invention is the first to discover that ligustilide can reduce the expression levels of S100A9 mRNA and protein in infarcted myocardial tissue and serum S100A9 expression levels in mice with myocardial infarction. It also found that ligustilide can inhibit the infiltration of neutrophils and monocytes in the infarcted area of ​​mice with myocardial infarction (MI), inhibit NETs formation after MI, and inhibit the expression and activation of NLRP3 inflammasomes. Therefore, ligustilide has both a preventive effect on the inflammatory response after myocardial ischemia and can alleviate the inflammatory response after MI, thereby protecting myocardial tissue. This invention also demonstrates through cardiac function testing, myocardial tissue staining, and serum biochemical testing that ligustilide can significantly improve cardiac function, downregulate serum CK, CK-MB, and LDH levels in the early stage of myocardial ischemia, and significantly reduce the number of apoptotic cardiomyocytes caused during MI injury, proving that ligustilide has a certain therapeutic effect on myocardial injury caused by MI. This invention expands the application scope of ligustilide in cardiovascular diseases and provides a new drug option for the prevention and treatment of inflammatory responses after myocardial ischemia, especially providing a new method for the prevention of inflammatory responses after myocardial ischemia. Attached Figure Description

[0017] Figure 1 These are the TTC staining results of the mouse hearts in each group in Example 1 of this invention; Figure A shows cross-sectional images of the myocardium after TTC staining in each group, and Figure B shows the statistical analysis of the percentage of myocardial infarction area in each group (n=6); compared with the Sham group, *** P <0.001; compared with the MI group, ### P <0.001, ## P <0.01, # P <0.05; Figure 2 These are representative echocardiogram images of mice from each group in Example 1 of this invention; Figure 3 This is a comparison of echocardiographic function in each group of mice in Example 1 of the present invention; compared with the Sham group, *** P <0.001; compared with the MI group, ### P <0.001, ## P <0.01, # P <0.05; Figure 4This is a comparison of the levels of CK, CK-MB, and LDH in the serum of mice in each group in Example 1 of this invention (n=4~6); compared with the Sham group, *** P <0.001; compared with the MI group, ### P <0.001, ## P <0.01, # P <0.05; Figure 5 These are the pathological damage changes in the myocardial tissue of mice in each group in Example 1 of this invention; Figure 6 This refers to the changes in cardiomyocyte apoptosis in the myocardial tissue of mice in each group in Example 1 of this invention; Figure 7 The effect of Lig on serum S100A9 levels after MI in Example 1 of this invention (n=4~6); compared with the Sham group, *** P <0.001; compared with the MI group, ## P <0.01, # P <0.05; Figure 8 This refers to the effect of Lig on S100A9 mRNA in cardiac tissue after MI in Example 1 of this invention (n=4~6); compared with the Sham group, *** P <0.001; compared with the MI group, ## P <0.01; Figure 9 This refers to the effect of Lig on S100A9 protein in cardiac tissue after MI in Example 1 of this invention (n=3); compared with the Sham group, ** P <0.01; compared with the MI group, # P <0.05; Figure 10 This refers to the effect of Lig on S100A9 expression in cardiac neutrophils after myocardial mitochondrial syndrome (n=4~6) in Example 1 of this invention; compared with the Sham group, * P <0.05; compared with the MI group, # P <0.05; Figure 11 This is the effect of Lig on the proportion and number of inflammatory cells in the heart after myocardial infarction (n=5~7) in Example 1 of this invention; Figure A shows the flow cytometry results of neutrophils and monocytes in the heart of mice in each group, and Figure B shows the quantitative graph of the proportion of neutrophils and monocytes to white blood cells in the heart of mice in each group; compared with the Sham group, *** P <0.001; compared with the MI group, ## P <0.01, # P <0.05; Figure 12This refers to the effect of Lig on serum dsDNA after MI in Example 1 of this invention (n=5~6); compared with the Sham group, *** P <0.001; compared with the MI group, # P <0.05; Figure 13 This refers to the effect of Lig on NETs-related proteins in myocardial tissue after myocardial MI in Example 1 of this invention (n=3); compared with the Sham group, *** P <0.001,** P <0.01; compared with the MI group, ## P <0.01, # P <0.05; Figure 14 This refers to the effect of Lig on the NLRP3 inflammasome pathway in Example 1 of this invention (n=3); compared with the Sham group, *** P <0.001,** P <0.01; compared with the MI group, ## P <0.01, # P <0.05; Figure 15 This refers to the effect of Lig on inflammatory factors in cardiac tissue after myocardial inflammatory disease (n=4~7) in Example 1 of this invention; compared with the Sham group, *** P <0.001; compared with the MI group, ### P <0.001, ## P <0.01, # P <0.05. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the implementation methods of this invention without inventive effort fall within the protection scope of this invention.

[0019] Myocardial ischemia triggers cardiac pathological inflammation. A persistently dysregulated inflammatory response exacerbates myocardial tissue damage, leading to myocardial ischemia and ultimately heart failure. S100A8 / A9 is rapidly released after ischemic injury, activating endothelial and immune cells, promoting the massive release of various inflammatory factors and chemokines, intensifying the local inflammatory response, causing further damage to cardiomyocytes, and inducing cardiomyocyte apoptosis. Elevated serum S100A8 / A9 levels are significantly associated with an increased risk of heart failure after acute myocardial infarction (MI) and are a key driver of the development and progression of heart failure. Regulating the inflammatory response is an effective means of intervening in MI and protecting against post-MI myocardial damage.

[0020] This invention, through research, has found that in the early stages of myocardial ischemia (MI), ligustilide significantly inhibits the expression and release of S100A9, reduces cardiac infiltration of neutrophils and monocytes, and inhibits neutrophil activation. Simultaneously, it downregulates the expression of MPO, PAD4, and H3cit proteins, reduces dsDNA release, and inhibits the formation of neutrophil extracellular traps (NETs). It also reduces the expression of NLRP3, ASC, and Caspase-1, inhibits the activation of the NLRP3 inflammasome, and reduces the production of inflammatory factors such as IL-1β and TNF-α in cardiac tissue. These effects collectively prevent and alleviate the inflammatory response following myocardial ischemia, ultimately protecting myocardial tissue. Based on these findings, this invention provides the application of ligustilide in the preparation of drugs for preventing or alleviating the inflammatory response following myocardial ischemia.

[0021] The following detailed description, using specific examples, will further illustrate this point.

[0022] The main reagents and consumables in the following examples are shown in Table 1.

[0023] Table 1 Experimental Reagents and Consumables

[0024] The main experimental instruments in the following embodiments are shown in Table 2.

[0025] Table 2 Experimental Instruments

[0026] Unless otherwise specified, all other reagents, pharmaceuticals, or instruments used in the following examples are commercially available products. The methods used in the following examples are conventional methods in the art.

[0027] Example 1 This embodiment provides the application of ligustilide in the preparation of drugs for preventing or reducing inflammatory responses following myocardial ischemia.

[0028] 1. Experimental Materials 1.1 Laboratory Animals SPF-grade C57BL / 6 male mice, weighing 22 ± 3 g, were purchased from Vital River (Beijing) Biotechnology Co., Ltd. The animal license number is SCXK (Beijing) 2019-0010. They were housed in the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine at a room temperature of 20-25°C and a relative humidity of 40%-60%. All operations on experimental animals were strictly carried out in accordance with the standards of the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC2019078).

[0029] 1.2 Experimental drugs Suxiao Jiuxin Pills (SJP) and ligustilide (Lig). SJP was purchased from the Sixth Traditional Chinese Medicine Factory of Tianjin Zhongxin Pharmaceutical Group Co., Ltd., with the product number C14003000981 and the approval number:国药准字Z12020025. Lig was purchased from Chengdu Dest Biotechnology Co., Ltd., with the product number 81944-09-4.

[0030] 1.3 Experimental reagents and consumables 1.4 Main solutions (1) Lig solution: Using corn oil as the solvent, Lig solutions with concentrations of 2 mg / mL, 4 mg / mL, and 6 mg / mL were prepared.

[0031] (2) SJP solution: Using normal saline as the solvent, an SJP solution with a concentration of 7.8 mg / mL was prepared and used immediately. The basis for the concentration of SJP is as follows: According to the clinical body surface area and equivalent dose conversion: The body weight of a person is 70 kg, and the adult dose is 600 mg / day; the body surface area ratio is 0.0026, and the equivalent dose ratio is 9.1; 9.1 / 70×600 mg = 78 mg. According to the administration dose for a 20 g mouse, it is 78 mg / kg.

[0032] (3) Picogreen fluorescence reaction solution: Dilute the Picogreen stock solution 200-fold with 1×TE buffer and prepare it immediately before use. Prepare and store it in the dark.

[0033] (4) Preparation of dsDNA standard product: The original concentration of the standard product is 100 μg / mL. Dilute it 50-fold with 1×TE buffer according to the required amount to obtain a working solution concentration of 2 μg / mL for standby.<( (5) Avertin (anesthetic): Accurately weigh 0.35 mg of tribromoethanol, dissolve it in 1 mL of tert-amyl alcohol, and heat it in a water bath at 65°C for 90 min. After it is completely dissolved, add 24 mL of double-distilled water and mix well. Filter it with a 0.22 μm filter membrane in the dark and then store it at 4°C.

[0034] (6) Enzyme digestion solution: Weigh 4 mg collagenase II (225 U / mg, 2 mg / mL) and 5 mg Dispase II (≥0.5 U / mg, 1.2 U / mL), and dissolve them in 2 mL of preheated mixture without calcium and magnesium and with added EDTA. The mixture consists of 1640 culture medium + 1% PS + 10 mM HEPES.

[0035] (7) Neutralization solution (FACS solution): PBS + 1% PS + 2% inactivated FBS + 2 mM EDTA.

[0036] (8) Cell culture medium: 1640 medium + 1% PS + 15% inactivated FBS.

[0037] 2. Experimental Methods 2.1 Preparation of a mouse model of myocardial ischemia Mice were acclimatized to a standard diet for one week, weighed, and randomly numbered according to their weight. They were then anesthetized with an intraperitoneal injection of Avertin (0.33 mL / 20 g). The mice were fixed in a supine position on the operating table, and the neck and chest were disinfected with iodine for skin preparation. A tracheal incision of approximately 5 mm was made at the trachea for intubation, followed by suturing. Under a dissecting microscope, transverse incisions were made at the axilla and midline of the xiphoid process. The skin was dissected, and visible capillaries were cauterized using a bipolar electrocoagulation device. The pectoralis major and pectoralis minor muscles were then separated and cauterized sequentially. A small animal ventilator was connected, and the ventilator parameters were set as follows: weight 0.05 kg, tidal volume 0.15 ccH2O, respiratory rate 145 BMP (breaths / min), and respiratory ratio 1:1. Using microscopic curved forceps, the third and fourth ribs were lifted. The muscles between the third and fourth ribs were cauterized using a bipolar electrocoagulation device. Two 6 / 0 monofilament nylon sutures were passed through the third and fourth ribs respectively. The suture at the third rib was fixed obliquely upwards, and the suture at the fourth rib was fixed vertically, fully exposing the heart. An 8 / 0 monofilament nylon suture was used to ligate the left atrial appendage to the apex at 1 / 3-1 / 2 of its length, leaving a distinct pale ischemic area below the ligation point. For Sham group mice, the thorax was opened, and a suture was passed through the same location as the coronary artery without ligation; all other procedures were the same. A flexible needle was inserted into the thoracic cavity for perfusion, and the pectoral muscle layers were sutured sequentially. Before removing the tube, the thoracic cavity was aspirated to negative pressure. After surgery, the animals were placed on a heated plate until they regained consciousness, and then placed in a clean cage. The success rate of the model was approximately 90%.

[0038] 2.2 Experimental Grouping Mice that successfully developed the model were randomly divided into 6 groups (see Table 3): Sham group, MI group, low-dose Lig group (Lig-L 20 mg / kg / d), medium-dose Lig group (Lig-M 40 mg / kg / d), high-dose Lig group (Lig-H 60 mg / kg / d), and SJP group (78 mg / kg / d). The first dose was administered 10 minutes post-surgery, followed by daily administration at the same time. The Sham group received an intraperitoneal injection of corn oil at a dose of 0.2 mL / 20 g body weight.

[0039] Table 3 Experimental Groups

[0040] 2.3 Cardiac function tests and cardiac tissue and blood sampling At 72 h post-surgery, the following parameters were measured in mice: left ventricular fractional shortening (LVFS), ejection fraction (LVEF), interventricular septal end-diastolic thickness (IVSd), interventricular septal end-systolic thickness (IVSs), left ventricular posterior wall thickness at end-diastole (LVPWd), left ventricular posterior wall thickness at end-systole (LVPWs), left ventricular end-diastolic diameter (LVIDd), and left ventricular end-systolic diameter (LVIDs). Mice were sedated with 0.2 mL / 20 g body weight of Avertin and placed in a supine position on a temperature-maintaining platform. Hair on the chest was removed with depilatory cream, and a heated ultrasonic transmissive gel was placed on the chest. Vevo 2100 was used for measurement. TM Doppler ultrasound was used to obtain clear two-dimensional images of the standard left ventricular region in a parasternal long-axis view. Left ventricular fractional shortening, ejection fraction, stroke volume, and cardiac output were assessed. M-mode echocardiography was used to acquire left ventricular systolic and diastolic motion curves at the level of the anterior and posterior leaflets of the mitral valve. Three cardiac cycles were averaged, and FS and EF were measured. In normal mice, EF% is generally ≥50%, and FS% is ≥30%. Generally, EF% ≤45% and FS% ≤30% are considered indicative of heart failure. Throughout the process, a heating platform and infrared heating lamps were used to maintain core body temperature at 37°C. Mice with a heart rate below 400 bpm (heart beats per minute) were excluded from the analysis.

[0041] 2.4 Changes in serum myocardial enzymes LDH, CK, and CK-MB were detected using a fully automated biochemical analyzer. At 24 h and 72 h post-surgery, 4–6 mice were randomly selected from each group. After anesthesia, blood was collected from the apex of the heart. The blood samples were left at room temperature for 2 h, then centrifuged at 4800 g for 20 min at 4°C. The supernatant was collected as serum. If red blood cells remained, the samples were centrifuged again at 4800 g for 10 min at 4°C, and the supernatant was collected as serum. After centrifugation, the serum samples were transferred to 1.5 mL EP tubes and stored at -80°C for later use. The levels of myocardial enzymes LDH, CK, and CK-MB in the undiluted serum were detected using a Hitachi 7020 fully automated biochemical analyzer.

[0042] 2.5 TTC staining to detect myocardial infarction area (1) 24 h after surgery, 6 mice were randomly selected from each group. After anesthesia, the heart was quickly removed, transferred in a 0-4℃ solution, and the heart tissue was frozen at -80℃ for 2 min.

[0043] (2) Frozen heart tissue sections (1 mm thick), about 4-5 sections per heart.

[0044] (3) Place the heart slices in the prepared 48-well plate, add 2% TTC staining solution, incubate at 37°C in the dark for 30 min, and gently shake once every 5 min to ensure thorough staining.

[0045] (4) Take out the stained section and observe the staining. The white area is the infarct area.

[0046] (5) Image J was used to analyze the infarct area, and GraphPad Prism 8.3.0 statistical analysis software was used for analysis.

[0047] 2.6 Paraffin embedding and sectioning At 24 h and 72 h post-surgery, one mouse from each group was randomly selected for intraperitoneal anesthesia with Avertin. The thoracic cavity was opened to expose the heart, and a perfusion needle was inserted from the apex of the heart into the aorta. The needle was fixed, the right atrial appendage was cut open, and the heart was rapidly perfused with PBS until the liver turned white and the outflow from the right atrial appendage became clear. Then, the heart was slowly perfused with 4% paraformaldehyde solution and thoroughly fixed before being harvested. The mouse heart was fixed in 4% paraformaldehyde for 48 h. After fixation, the heart was removed and cut with a scalpel to facilitate the penetration of alcohol, xylene, and paraffin. The heart was then dehydrated, cleared, infiltrated with paraffin, and embedded sequentially to prepare 4-micrometer-thick paraffin sections for HE staining and TUNEL staining.

[0048] 2.7 HE staining for myocardial histopathology Paraffin sections of myocardial tissue 3 days after surgery were stained using hematoxylin-eosin staining (HE) and observed and photographed under a microscope.

[0049] 2.8 TUNEL staining for cardiomyocyte apoptosis in myocardial tissue Paraffin sections of myocardial tissue taken 1 day (24 h) postoperatively were stained using the TUNEL staining method and observed under a fluorescence microscope.

[0050] 2.9 Flow cytometry detection of inflammatory cell changes and S100A9 expression in infarcted cardiac tissue (1) 24 h postoperatively, 5-7 mice were randomly selected from each group and their hearts were perfused with pre-cooled Hanks until the lungs and liver turned white and the perfusion fluid was clear. Using the ligation point as a marker, infarct tissue was cut from the heart of each mouse and placed in a 2.0 mL centrifuge tube containing 1 mL of pre-cooled PBS.

[0051] (2) The myocardial tissue was thoroughly shredded on ice and centrifuged at 4°C, 50 g for 1 min.

[0052] (3) Discard the supernatant, add the enzyme digestion solution, place in a metal bath at 37°C for 30 min, and mix once every 10 min.

[0053] (4) Transfer the tissue to a C tube and run the “mouse_heart_01” program in Gentle MACS. After the program is complete, add 10 mL of pre-cooled neutralization solution, filter through a 70 μm filter membrane, and then centrifuge at 350 g for 5 min at 4 °C.

[0054] (5) Collect cells, centrifuge at 350 g for 5 min, remove the supernatant, add 1 mL PBS, and discard the supernatant.

[0055] (6) Completely thaw the Cell Activation Cocktail (containing Brefeldin A) in a 37°C water bath. Resuspend the cells in cell culture medium. For every 1×10⁶ cells... 6 Add 2 μL of Cocktail to the cell suspension (cells / mL) and incubate in a 37℃ CO2 incubator for 4 h. A naked cell control group is also set up.

[0056] (7) Collect cells, centrifuge at 350 g for 5 min at 4℃, and discard the supernatant.

[0057] (8) Centrifuge at 350 g for 5 min at 4℃, wash once with 1 mL PBS to remove the supernatant.

[0058] (9) Add 1 μL of flow cytometry antibody to each FACS solution in the dark, gently vortex to mix, place the sample on ice and incubate for 30 min in the dark.

[0059] (10) Resuspend the cells in 1 mL of 1× membrane-breaking agent, centrifuge at 350 g for 10 min at 4°C, remove the supernatant, and repeat this step once.

[0060] (11) Add intracellular antibody to 1× membrane permeabilizing agent, gently vortex mix for 3 s, and then incubate at room temperature in the dark for 20 min.

[0061] (12) Resuspend the cells in 2 mL of 1× intracellular staining permeabilization washing buffer, centrifuge at 4℃ and 350 g for 5 min, remove the supernatant, and repeat this step once.

[0062] (13) Resuspend in 200 µL of Permeabilization Wash Buffer and test on the machine.

[0063] 2.10 Western Blot Detection Twenty-four hours after surgery, four to six mice were randomly selected from each group. After anesthesia, blood was collected from the apex of the heart, and the heart tissue was perfused with 1×PBS solution. Then, the infarcted area of ​​the heart was excised using the ligation point as a marker, and total protein was extracted. Western blotting was used to detect the protein expression levels of S100A9, MPO, PAD4 and H3cit, as well as NLRP3, ASC, Pro-caspase-1 and Cleaved-caspase-1.

[0064] 2.11 Reverse transcriptase polymerase chain reaction (RT-qPCR) Twenty-four hours post-surgery, three mice were randomly selected from each group. After anesthesia, cardiac tissue was perfused with 1×PBS solution. The infarcted area was then excised using the ligation point as a marker. The sample was frozen to ultra-low temperature and then rapidly transferred to a mortar pre-cooled with liquid nitrogen. It was thoroughly ground with a pestle until it became powder, and RNA was extracted using standard methods. The purity and concentration of the RNA sample were determined, followed by reverse transcription. RT-qPCR was performed on a Real-Time PCR instrument using the TransStart® Green qPCR SuperMix kit to detect gene expression in each sample. The Ct value for each sample was obtained, and the ΔCt was calculated using a relative quantification method: ΔCt = Ct target gene - Ct internal reference gene. After comparison with the control group, two samples were taken. -ΔΔCtThe values ​​are used for data statistics.

[0065] Table 8 Primer sequence list

[0066] 2.12 Detection of S100A9 content in serum by ELISA (1) 24 hours after the operation, 4 to 6 mice were randomly selected from each group. After anesthesia, blood was taken from the apex of the heart and the blood samples were processed according to the method in “2.4” to obtain serum.

[0067] (2) Add the standard working solution sequentially to the first two rows of wells, with two wells of each concentration added side-by-side, 100 μL per well. Add the serum to be tested to the other wells, 100 μL per well. Cover the microplate with a membrane and incubate at 37°C for 90 min. Add the sample to the bottom of the microplate, avoiding contact with the well walls as much as possible, and gently shake to mix, avoiding the formation of air bubbles.

[0068] (3) Discard the liquid, shake dry, and do not wash. Add 100 μL of biotinylated antibody working solution to each well, mix well, cover the plate with a membrane, and incubate at 37°C for 1 h.

[0069] (4) Shake off the liquid in the wells, add 350 μL of washing solution to each well, soak for 1-2 min, aspirate or shake off the liquid in the microplate, pat dry on absorbent paper, and repeat this step 3 times.

[0070] (5) Add 100 μL of enzyme conjugate working solution to each well, cover with a membrane, and incubate at 37°C for 30 min.

[0071] (6) Discard the liquid in the hole, spin dry, and wash the plate 5 times.

[0072] (7) Add 90 μL of substrate solution (TMB) to each well, cover the microplate with a membrane, and incubate at 37°C in the dark for 15 min.

[0073] (8) Add 50 μL of stop solution to each well to terminate the reaction. The order of adding the stop solution is the same as the order of adding the substrate solution.

[0074] Immediately measure the optical density (OD value) of each well using an ELISA reader at a wavelength of 450 nm.

[0075] 2.13 PicoGreen TM dsDNA detection (1) 24 hours after the operation, 5 to 6 mice were randomly selected from each group. After anesthesia, blood was taken from the apex of the heart and the blood samples were processed according to the method in “2.4” to obtain serum.

[0076] (2) Preparation of standard curve: Take 5 sterile 1.5 mL EP tubes and add 100 μL, 10 μL, 1 μL, 0.1 μL and 0 μL of dsDNA standard (DNA concentration 2 μg / mL) to the EP tubes respectively. Then add 0 μL, 90 μL, 99 μL, 99.9 μL and 100 μL of 1×TE buffer to the EP tubes respectively. (3) Add 5 standards to a sterile black transparent 96-well cell culture plate in sequence at a rate of 100 μL / well using a sterile pipette tip.

[0077] (4) After adding the standard, add the serum to be tested (processing method is the same as "2.4") starting from the second column, 100 μL / well. After the sample addition is completed, add the pre-prepared Picogreen fluorescent reaction solution to the standard wells and the serum wells, 100 μL / well. After adding, protect from light and gently shake horizontally for 1 min.

[0078] (5) Place the black 96-well plate into a multi-functional microplate reader at a wavelength of 480 / 520 nm and scan the plate to detect the OD values ​​of the standards and samples.

[0079] (6) Substitute the OD value into the standard curve to obtain the corresponding NET-DNA concentration.

[0080] 2.14 Statistical Analysis All data are expressed as mean ± standard deviation (mean ± SD). The Student's-test was used to compare the two groups. GraphPadPrism 8.3.0 statistical analysis software was used for analysis. Independent samples t-test was used to compare the two groups. One-way ANOVA was used to compare the data between the two groups. P < 0.05 was considered statistically significant.

[0081] 3. Experimental Results 3.1 Experimental results of the anti-myocardial ischemia injury effect of Lig 3.1.1 Lig reduces the infarct area of ​​myocardial tissue after MI injury Infarct size is a key indicator for assessing the severity of myocardial infarction (MI). TTC staining was used to stain the heart tissue of mice in each group 24 hours post-surgery, and the degree of myocardial infarction was assessed by observing the color differences in myocardial tissue. Red represents normal myocardial tissue, while infarcted tissue appears white due to lack of activity. Results are as follows: Figure 1As shown, the cardiac tissue in the sham group was uniformly red, while the myocardial tissue in the model (MI) group exhibited large areas of white infarcts, indicating a significantly increased area of ​​myocardial necrosis. Compared to the MI group, the infarct area in the Lig group and the positive control (SJP) group was significantly reduced, and the infarct area in the Lig group decreased gradually with increasing drug concentration. These results suggest that Lig, within a certain dose range, has a dose-gradient effect in reducing the infarct area in MI mice.

[0082] 3.1.2 Lig significantly improved cardiac function in MI mice Echocardiogram images as follows Figure 2 As shown, the results of the cardiac function index tests are as follows: Figure 3 As shown in the figure. Compared with the Sham group, the MI group showed significantly decreased LVEF, LVFS, and LVPWs postoperatively, and significantly increased LVIDd, indicating severe heart failure in mice 3 days after myocardial ischemia. Compared with the MI group, Lig improved LVEF, LVFS, IVSs, LVPWd, LVPWs, and LVIDd. These results suggest that Lig can improve cardiac function by enhancing ventricular diastolic and systolic function.

[0083] 3.1.3 Lig significantly reduced serum levels of CK, CK-MB, and LDH. CK, CK-MB and LDH levels as follows Figure 4 As shown, compared with the Sham group, the levels of CK, CK-MB, and LDH in the MI group were significantly increased, indicating that cardiomyocytes were damaged after MI. After 1 day and 3 days of administration, compared with the MI group, the serum levels of CK, CK-MB, and LDH in the Lig group and the positive control drug SJP group were significantly reduced, and overall, the high-dose Lig showed the best downregulation effect. The results indicate that Lig and SJP have a certain protective effect against myocardial injury in the early stage of myocardial ischemia.

[0084] 3.1.4 Lig reduces myocardial tissue pathological damage after MI injury The pathological changes in the heart tissue of mice in each group are as follows: Figure 5 As shown, the Sham group exhibited clear cardiac tissue structure, with tightly and orderly arranged myocardial fibers and no obvious pathological changes. Compared to the Sham group, the MI group showed loose cardiac tissue structure, disordered myocardial cell arrangement, indistinct myocardial fiber striations, and extensive inflammatory cell infiltration around the infarct area. Compared to the MI group, the Lig group and the positive control drug SJP group showed improved myocardial fiber arrangement, significantly reduced breakage and dissolution, and decreased inflammatory cell infiltration. These results suggest that Lig has a certain therapeutic effect on myocardial damage caused by MI and can improve the pathological state of myocardial tissue.

[0085] 3.1.5 Lig reduces cardiomyocyte apoptosis in myocardial tissue after myocardial injury. Apoptosis of myocardial tissue, such as Figure 6 As shown, compared with the Sham group, the number of apoptotic cardiomyocytes in the infarcted area of ​​the heart tissue was significantly increased in the MI group; compared with the MI group, the number of apoptotic cardiomyocytes in the infarcted area of ​​the heart tissue was significantly reduced in the Lig-H group and the SJP group. The results indicate that Lig and SJP have an ameliorative effect on cardiomyocyte apoptosis induced during MI injury.

[0086] 3.2. Lig administration reduced the expression of S100A9 mRNA and protein in infarcted myocardial tissue and the serum S100A9 level in mice with myocardial infarction. Following myocardial infarction (MI), S100A9 is rapidly released as an early DAMP. S100A9 can serve as a biomarker for MI, and elevated serum levels can indicate the occurrence of MI. S100A9 levels are also closely related to poor prognosis after MI; high S100A9 levels predict a higher risk of heart failure and cardiovascular events. In this experiment, the expression level of S100A9 in the serum of mice in each group was detected by ELISA 24 h after MI. The results are as follows: Figure 7 As shown, compared with the Sham group, the serum S100A9 level in the MI group was significantly increased; 24 h after administration, compared with the MI group, Lig and SJP significantly inhibited the serum S100A9 level.

[0087] To further demonstrate the inhibitory effects of Lig and SJP on S100A9 after myocardial infarction (MI), RT-qPCR and Western blot were used to detect the changes in S100A9 mRNA and protein expression in the infarcted area after MI. The results are as follows: Figure 8 , Figure 9 As shown, compared with the Sham group, the expression of S100A9 mRNA and protein in the myocardial infarction area was significantly upregulated in the MI group; 24 h after administration, compared with the MI group, Lig and SJP significantly inhibited the expression of S100A9 mRNA and protein in the myocardial infarction area.

[0088] Studies have shown that S100A9 is highly expressed in neutrophils and is an important component of neutrophils. Therefore, flow cytometry was used to detect S100A9 expression in neutrophils in the myocardial infarction area, and the results are as follows: Figure 10 As shown, compared with the Sham group, the expression of S100A9 in neutrophils in the myocardial infarction area was significantly upregulated in the MI group; 24 h after administration, compared with the MI group, Lig significantly inhibited the expression of S100A9 in neutrophils in the myocardial infarction area, and SJP showed a trend of reducing its expression, but it was not statistically significant.

[0089] In summary, these results suggest that in the early stages of myocardial ischemia, Lig and SJP inhibit the infiltration of inflammatory cells, especially neutrophils and monocytes, thereby suppressing the inflammatory response, reducing the release of S100A9, and thus lowering the level of S100A9, thereby playing a protective role against myocardial injury.

[0090] 3.3 Lig intervention inhibits neutrophil and monocyte infiltration in the infarct area of ​​MI mice Within 24 hours after myocardial ischemia, neutrophils are the first inflammatory cells to infiltrate the ischemic area. Following neutrophil infiltration, monocytes also begin to enter the ischemic area. The infiltration of both neutrophils and monocytes typically peaks around 24 hours after myocardial ischemia. A moderate inflammatory response is beneficial for myocardial repair, but an excessive inflammatory response may lead to further damage to cardiomyocytes. Therefore, 24 hours after myocardial ischemia, flow cytometry was used to detect the proportion and number of neutrophils and monocytes in the ischemic myocardial tissue of mice in each group. The results are as follows: Figure 11 As shown, compared with the Sham group, the proportion of neutrophils and monocytes in the white blood cell count of ischemic heart tissue was significantly increased in the MI group mice, indicating that the MI model successfully induced an inflammatory response in the heart tissue, leading to increased infiltration of neutrophils and monocytes. 24 h after administration, compared with the MI group, the proportion of neutrophils and monocytes in the white blood cell count was significantly decreased in the Lig-H and SJP groups. These results suggest that in the early stages of myocardial ischemia, Lig and SJP can inhibit the inflammatory response in heart tissue by reducing the infiltration of neutrophils and monocytes.

[0091] 3.4 Lig inhibits NETs formation and NLRP3 inflammasome expression and activation after MI 3.4.1 Lig inhibits NET formation in cardiac tissues after myocardial infarction (MI). The formation of neutrophil NETs is an important marker of inflammatory damage following myocardial ischemia. This study assessed NET levels by detecting serum cell-free double-stranded DNA (dsDNA) content. The results are as follows: Figure 12 As shown, compared with the Sham group, the serum dsDNA content of mice in the MI group was significantly increased, indicating that neutrophils were overactivated and released a large number of NETs after MI; 24 h after administration, the serum dsDNA level in the Lig intervention group was significantly lower than that in the MI group, indicating that Lig can effectively inhibit NET formation.

[0092] Following myocardial infarction (MI), neutrophils are rapidly recruited to the infarcted cardiac tissue, releasing large amounts of NETs to participate in the inflammatory response and tissue damage. MPO and PAD4 are key enzymes in NET formation, while H3cit is a biomarker for NET formation. Therefore, 24 h after MI, Western blot was used to detect the effects of Lig and SJP on neutrophil NET formation-related proteins MPO, PAD4, and H3cit. The results are as follows: Figure 13 As shown, compared with the Sham group, the expression of MPO, PAD4, and H3cit proteins in the infarcted cardiac tissue of the MI group was significantly upregulated, indicating that neutrophils were activated in the infarcted cardiac tissue and released NETs. 24 h after drug administration, compared with the MI group, Lig and SJP significantly downregulated the expression of MPO and PAD4, inhibiting NET formation; the downregulation of H3cit further confirmed the reduction in NET formation after drug intervention. These results suggest that Lig and SJP may protect damaged myocardial tissue by inhibiting the formation of neutrophil NETs.

[0093] 3.4.2 Lig's effect on NLRP3 inflammasome expression and activation after MI Twenty-four hours after myocardial infarction (MI), Western blot and RT-qPCR were used to detect the effects of Lig and SJP on the NLRP3 inflammasome pathway in the infarcted area after MI. Results are as follows: Figure 14 As shown, compared with the Sham group, the protein expression of NLRP3, ASC, Pro-caspase-1, and Cleaved-caspase-1 in the infarcted cardiac tissue of the MI group was significantly upregulated, indicating that myocardial infarction activates the NLRP3 inflammasome pathway. 24 h after administration, compared with the MI group, Lig and SJP significantly downregulated the protein expression of NLRP3 and ASC, and the protein expression of Pro-caspase-1 and Cleaved-caspase-1 showed a downregulation trend, but this was not statistically significant. RT-qPCR results showed that compared with the Sham group, the mRNA expression of NLRP3 and Caspase-1 in the infarcted cardiac tissue of the MI group was significantly upregulated; 24 h after administration, compared with the MI group, Lig and SJP significantly downregulated the mRNA expression of NLRP3 and Caspase-1. These results suggest that Lig and SJP may indirectly reduce Caspase-1 activation and alleviate the inflammatory response after MI by inhibiting the assembly of the NLRP3 inflammasome.

[0094] 3.4.3 Effects of Lig on cytokines in cardiac tissue of MI mice 24 hours after myocardial infarction (MI), RT-qPCR was used to detect the mRNA expression of IL-1β and TNF-α in the infarcted area of ​​cardiac tissue to further observe the effect of IL-1β on the inflammatory response of cardiac tissue. Results are as follows: Figure 15 As shown, compared with the Sham group, the mRNA expression of IL-1β and TNF-α in the myocardial infarction area of ​​mice in the MI group was significantly increased. 24 h after administration, compared with the MI group, Lig and SJP significantly inhibited the mRNA expression of IL-1β and TNF-α. These results suggest that Lig and SJP may protect myocardial tissue by inhibiting the expression of these inflammatory factors and thus alleviating the inflammatory response.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of ligustilide in the preparation of drugs for preventing or reducing inflammatory responses after myocardial ischemia, characterized in that, The functional components of the drug include ligustilide.

2. The application according to claim 1, characterized in that, The myocardial ischemia mentioned refers to acute myocardial ischemia.

3. The application according to claim 1, characterized in that, The drug in question is for preventing inflammatory responses following myocardial ischemia.

4. The application according to claim 1, characterized in that, The drug is a drug that inhibits the level of S100A9 in serum.

5. The application according to claim 1, characterized in that, The drug is a drug that inhibits the expression of S100A9 mRNA and protein in the myocardial infarction zone.

6. The application according to claim 1, characterized in that, The drug is a drug that inhibits the expression of S100A9 in neutrophils.

7. The application according to any one of claims 1 to 6, characterized in that, The drug is an oral formulation.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

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