3-hppa for use in the preparation of a medicament for the prevention or treatment of myocardial infarction

Abstract

The application discloses a new application of 3-HPPA in preparing a drug for treating myocardial infarction. By means of modern pharmacological research methods, it is first confirmed that 3-HPPA has a significant myocardial protective effect. Experimental research shows that 3-HPPA can significantly improve the heart function of a myocardial infarction mouse induced by coronary artery ligation, improve the left ventricular ejection fraction and the short axis shortening rate, and reduce the infarction area; meanwhile, 3-HPPA can significantly inhibit the gene expression of a heart failure marker BNP, and down-regulate the expression levels of inflammatory factors IL-6, TNF-alpha and fibrosis related factors TGF-beta and Collagen I. Further mechanism research shows that 3-HPPA also plays an anti-inflammatory, anti-fibrosis and anti-apoptosis role. The application discloses the action characteristics of 3-HPPA in multi-target intervention of the pathological process of myocardial infarction, provides an important candidate compound for development of a new type of anti-myocardial infarction drug, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of 3-HPPA in the preparation of drugs for the prevention or treatment of myocardial infarction. Background Technology

[0002] Myocardial infarction (MI) is one of the leading causes of death and disability among cardiovascular diseases worldwide. According to the World Health Organization (WHO), cardiovascular diseases (CVDs) cause approximately 17.9 million deaths annually, with myocardial infarction accounting for over 40% of these deaths. In my country, with an aging population and the high incidence of metabolic diseases such as hypertension and diabetes, the incidence of myocardial infarction is increasing year by year, and the age of onset is gradually becoming younger. The proportion of myocardial infarction patients under the age of 45 has risen from 5% in 2000 to 12% currently, and many present with sudden death as the initial symptom, severely impacting the workforce. These characteristics make the prevention and treatment of myocardial infarction a major issue in my country's public health field, urgently requiring the development of more effective treatment strategies.

[0003] Currently, clinical treatment for myocardial infarction mainly includes reperfusion therapy (such as thrombolysis and percutaneous coronary intervention (PCI)), antiplatelet drugs (such as aspirin and clopidogrel), beta-blockers, and ACE inhibitors. While these methods can alleviate symptoms to some extent, they still have many limitations. For example, reperfusion therapy has a limited "time window," and many patients miss the optimal treatment opportunity due to delayed medical attention; long-term use of antiplatelet drugs may increase the risk of bleeding; and existing drugs cannot completely reverse myocardial fibrosis and ventricular remodeling, leading to adverse outcomes such as heart failure. Therefore, developing novel cardioprotective drugs, especially natural active ingredients that can inhibit inflammatory responses, reduce cardiomyocyte apoptosis, and improve cardiac remodeling, is of significant clinical importance.

[0004] The core pathological process of myocardial infarction is acute coronary artery occlusion leading to myocardial ischemia and hypoxia, which in turn triggers a series of cascade reactions, including inflammatory response and myocardial injury. After myocardial ischemia, necrotic cells release damage-associated molecular patterns (DAMPs), activate Toll-like receptors (TLRs) and the NF-κB pathway, promote macrophage infiltration, and release pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, exacerbating myocardial injury. Inhibiting excessive inflammatory response can reduce infarct size and improve cardiac function. Oxidative stress and apoptosis: Ischemia-reperfusion (I / R) injury leads to a large accumulation of reactive oxygen species (ROS), which damages mitochondrial function, activates the Bax / Bcl-2-Caspase-3 apoptosis pathway, and leads to cardiomyocyte death. Therefore, anti-oxidation and anti-apoptosis strategies (such as regulating the Nrf2 / HO-1 pathway) are potential therapeutic directions. Myocardial fibrosis and ventricular remodeling: After myocardial infarction, cardiac fibroblasts are activated and secrete large amounts of collagen (Collagen I / III), leading to increased myocardial stiffness and eventually heart failure. The TGF-β / Smad and Wnt / β-catenin signaling pathways play a key regulatory role in this process, and inhibiting these pathways can slow down the fibrosis process.

[0005] Currently used anti-myocardial infarction drugs, such as antiplatelet drugs, reduce myocardial oxygen consumption by inhibiting thrombus formation, β-blockers, and ACEI / ARB improve ventricular remodeling, have problems such as single target, difficulty in comprehensively intervening in the complex pathological process of myocardial infarction, limited repair effect on existing myocardial damage, and potential adverse reactions with long-term use.

[0006] Traditional Chinese medicine (TCM) has the characteristics of multi-component, multi-target, and holistic regulation in the treatment of cardiovascular diseases, especially in improving microcirculation, inhibiting inflammation, and promoting tissue repair. For example, TCM herbs that promote blood circulation and remove blood stasis (such as Danshen and Sanqi) can improve myocardial microcirculation; TCM herbs that nourish qi and yin (such as ginseng and Ophiopogon japonicus) can reduce myocardial cell apoptosis; and TCM herbs that clear heat and detoxify (such as Coptis chinensis and Scutellaria baicalensis) have anti-inflammatory effects.

[0007] The Guanxin No. 1 Formula is a commonly used clinical compound for promoting blood circulation and removing blood stasis, composed of ingredients such as Danshen, Sanqi, and Hongjingtian. Previous studies have confirmed that it can improve myocardial blood supply and reduce infarct area. However, its specific pharmacodynamic material basis and mechanism of action are still unclear, limiting the modern development and clinical application of this formula. In recent years, the development of serum pharmacochemistry and metabolomics technologies has provided new ideas for the research of traditional Chinese medicine compound formulas. By analyzing the original components and metabolites in drug-containing serum, the active substances that truly exert the pharmacological effect can be screened out. This study found that 3-HPPA (3-(3-hydroxyphenyl)propionic acid) is a key blood-entering component of the Guanxin No. 1 Formula, and its concentration in drug-containing serum is significantly higher than that in blank serum, suggesting that it may be the core pharmacodynamic substance. Existing studies have shown that 3-HPPA has significant anti-inflammatory effects, reducing the levels of inflammatory factors such as IL-6 and TNF-α; it has clear antioxidant activity, reducing oxidative damage by scavenging ROS; and it has potential cardiovascular protective effects, with literature reporting that it can dilate blood vessels through the NO pathway. However, research on the application of 3-HPPA in myocardial infarction is still lacking, especially its regulation of the Wnt / β-catenin signaling pathway, its inhibitory effect on myocardial fibrosis, and its intervention mechanism for apoptosis. Summary of the Invention

[0008] The purpose of this invention is to provide a novel use of 3-(3-hydroxyphenyl)propionic acid (3-HPPA) in the treatment of myocardial infarction.

[0009] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: A first aspect of the present invention provides the use of 3-HPPA in the preparation of medicaments for the prevention and / or treatment of myocardial infarction, wherein the structural formula of the 3-HPPA is shown in formula (I): .

[0010] In some embodiments of the present invention, the myocardial infarction includes ST-segment elevation myocardial infarction.

[0011] In some embodiments of the invention, the 3-HPPA comprises a pharmaceutically acceptable salt.

[0012] In some embodiments of the present invention, the pharmaceutically acceptable salt includes an alkali addition salt.

[0013] In some embodiments of the present invention, a "pharmaceutically acceptable base addition salt" refers to a salt that retains the biological effectiveness and properties of the free acid and is not undesirable in biological or other respects. These salts are prepared by the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Preferred inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, the following: primary, secondary, and tertiary amines; substituted amines (including naturally occurring substituted amines); cyclic amines; and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, thiazoline, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0014] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0015] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, release inhibitors, and carriers.

[0016] Furthermore, to facilitate medication, the active ingredient 3-HPPA can be formulated into a specific dosage form with one or more pharmaceutically acceptable excipients. These excipients can be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, and microcrystalline cellulose), absorbents (e.g., calcium sulfate, dicalcium phosphate, light magnesium oxide, and calcium carbonate), wetting agents (e.g., water and ethanol), binders (e.g., hydroxypropyl methylcellulose, povidone, starch paste, and syrup), disintegrants (e.g., dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone), and lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized powders). The following are examples of agents: silica gel, colorants (such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide), coating materials (such as acrylic resin, hydroxypropyl methylcellulose, and povidone), solvents (such as water for injection, ethanol, propylene glycol, and glycerin), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, and sodium tartrate), antioxidants (such as sodium sulfite, sodium metabisulfite, and sodium thiosulfate), antibacterial agents (such as phenol, benzyl alcohol, and thimerosal), and isotonic regulators (such as sodium chloride and glucose).

[0017] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0018] In some embodiments of the present invention, the dosage form of the drug includes tablets, capsules, granules, injections, oral liquids, or sustained-release formulations.

[0019] In some embodiments of the present invention, the drug is administered to mammals; preferably, the mammals include humans.

[0020] In some embodiments of the present invention, the drug further includes any one or more other active ingredients.

[0021] In some embodiments of the present invention, the other active ingredients have the function of treating myocardial infarction.

[0022] In some embodiments of the present invention, the other active ingredients include, but are not limited to, tenepase, aspirin, ticagrelor, statins, etc.

[0023] In some embodiments of the present invention, the drug has one or more of the following effects: 1) Improves cardiac contractile function after myocardial infarction; 2) Reduce the area of ​​myocardial infarction; 3) Inhibits the gene expression level of BNP in myocardial tissue; 4) Reduce the expression levels of inflammatory factors IL-6 and TNF-α in myocardial tissue; 5) Inhibit the expression levels of myocardial fibrosis-related factors TGF-β and Collagen I; 6) It exerts a cardioprotective effect by inhibiting the Wnt2 / β-catenin signaling pathway.

[0024] The beneficial effects of this invention are: This invention discloses for the first time a novel pharmaceutical use of 3-HPPA in the prevention and / or treatment of myocardial infarction, and confirms its significant cardioprotective effect through systematic experimental studies. The innovation and beneficial effects of this invention are mainly reflected in its ability to significantly improve cardiac function. Animal experiments have shown that 3-HPPA treatment can significantly increase the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS) in mice with myocardial infarction, improve cardiac contractile function, reduce the degree of myocardial fibrosis, and protect the structural integrity of myocardial tissue. It can also regulate pathological processes at multiple targets, such as anti-inflammatory effects: significantly inhibiting the expression of pro-inflammatory factors IL-6 and TNF-α, reducing myocardial inflammatory response; anti-fibrotic effects: downregulating the expression of TGF-β and Collagen I, improving myocardial remodeling; anti-apoptotic effects: regulating the Bax / Bcl-2 balance, inhibiting Caspase-3 activation, and reducing cardiomyocyte apoptosis; and for the first time, it has been revealed that 3-HPPA exerts its cardioprotective effect by inhibiting the Wnt2 / β-catenin signaling pathway, providing a new target for the treatment of myocardial infarction. Furthermore, the pharmacodynamic material basis is clearly defined: metabolomics studies have confirmed that 3-HPPA is the key active ingredient in the efficacy of the Guanxin No. 1 formula in vivo, and its concentration in drug-containing serum is significantly higher than that in blank serum, providing a scientific basis for the modernization of traditional Chinese medicine compound research. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Effects of different concentrations of 3-HPPA on cardiac function in MI mice; (A) Left ventricular ejection fraction (EF) of mice in each group; (B) Left ventricular shortening fraction (FS) of mice in each group.

[0026] Figure 2 The effects of different concentrations of 3-HPPA treatment on organ coefficients and postoperative survival rates in MI mice were investigated. (A) Heart weight to body weight ratio (HW / BW) of mice in each group, (B) Heart weight to tibia length ratio (HW / TL) of mice in each group, and (C) Survival curves of mice in each group.

[0027] Figure 3 The effects of different concentrations of 3-HPPA treatment on serum inflammatory factors in MI mice; (A) IL-6 (interleukin-6) levels in each group of mice; (B) IL-1β (interleukin-1β) levels in each group of mice. Figure 4 To investigate the effects of different concentrations of 3-HPPA treatment on myocardial infarction in MI mice, TTC staining was performed on heart tissue sections from each group of mice.

[0028] Figure 5 To investigate the effects of different concentrations of 3-HPPA treatment on myocardial injury markers in MI mice, the following data were used: (A) expression level of myocardial injury-related gene ANP, (B) expression level of myocardial injury-related gene BNP, and (C) expression level of myocardial injury-related gene MYH7.

[0029] Figure 6 The effects of different concentrations of 3-HPPA treatment on inflammatory factors in myocardial tissue of MI mice were investigated. (A) Changes in the content of myocardial inflammatory factor IL-1β, (B) Changes in the content of myocardial inflammatory factor IL-6, and (C) Changes in the content of myocardial inflammatory factor TNF-α.

[0030] Figure 7 To investigate the effects of different concentrations of 3-HPPA treatment on apoptosis in the myocardial tissue of MI mice, (A) mRNA levels of apoptosis factor Bax, (B) mRNA levels of anti-apoptotic factor Bcl2, and (C) mRNA levels of Caspase-3.

[0031] Figure 8 The effects of different concentrations of 3-HPPA treatment on inflammatory and fibrotic factors in MI mice: (A) NF-κB expression level, (B) Collagen I expression level. Detailed Implementation

[0032] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0033] This invention utilizes left anterior descending coronary artery ligation (LAD) to establish a C57BL / 6 mouse model of myocardial infarction, revealing for the first time the significant therapeutic effects of 3 mg / mL and 9 mg / mL 3-HPPA in treating myocardial infarction. Mice were randomly divided into six groups: sham-operated group (Sham), model group (MI), low-dose 3-HPPA group (3 mg / mL), high-dose 3-HPPA group (9 mg / mL), the "Coronary Heart No. 1 Formula" treatment group (GXYHF, 11.05 g / kg), and the captopril positive control group (KTPL, 10 mg / kg). Mice were administered the medication continuously for 4 weeks post-surgery, and their body weight, cardiac function, and other indicators were monitored regularly. Experimental results showed that all 3-HPPA dose groups significantly improved cardiac function compared to the model group. The 3 mg / mL group showed a significant increase in left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS). 3-HPPA treatment significantly reduced myocardial infarction area, with the high-dose group showing a significant reduction in infarct area compared to the model group. qPCR analysis showed that 3-HPPA dose-dependently inhibited the mRNA expression of heart failure markers such as BNP and ANP in myocardial tissue, and significantly downregulated the protein expression of inflammatory factors IL-6 and TNF-α, and fibrosis markers Collagen I and TGF-β. Furthermore, 3-HPPA reduced cardiomyocyte apoptosis by inhibiting the mRNA expression of the Wnt2 / β-catenin pathway. The 3-HPPA treatment group showed a significant reduction in myocardial fibrosis, with myocardial tissue structure closer to normal, significantly improving cardiac function and ventricular remodeling after myocardial infarction. This study demonstrates that 3-HPPA works through a multi-target mechanism: improving myocardial ischemia and hypoxia, inhibiting excessive inflammatory response, reducing myocardial fibrosis, and decreasing cardiomyocyte apoptosis. This indicates that 3-HPPA can significantly treat myocardial infarction with low toxicity and no metabolic inhibition, providing a novel candidate drug for the clinical treatment of myocardial infarction. It has significant clinical application value and offers a new direction for the treatment of myocardial infarction.

[0034] The relevant material information for this invention is as follows: Experimental animals: 120 male C57BL / 6J mice, 8 weeks old, weighing 18-22 g, provided by Zhuhai Baishitong Biotechnology Co., Ltd.

[0035] Test drug: 3-(3-hydroxyphenyl)propionic acid (3-HPPA), purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0036] Maintenance feed: Purchased from Keao Cooperation (Tianjin) Feed Co., Ltd.

[0037] All animal experiments were conducted in strict accordance with the requirements of the "Guidelines for the Management and Use of Laboratory Animals" and with the permission of the Laboratory Animal Management Committee of the Zhongshan Institute of Drug Innovation, Chinese Academy of Sciences. During animal sacrifice, animals were anesthetized, and their suffering was minimized as much as possible.

[0038] Example I. Experimental Methods 1. Mouse grouping and drug administration One hundred and twenty healthy male C57BL / 6 mice were acclimatized for one week (hospitalization conditions: ambient temperature 22±0.5℃, 12-hour / 12-hour light-dark alternation). The animals were randomly divided into six groups: sham-operated group, model group, low-dose 3-HPPA group, high-dose 3-HPPA group, Coronary Heart Disease Formula No. 1 treatment group, and captopril positive control group.

[0039] (1) Sham surgery group (Sham, n=20): The chest was opened to the heart and sutured, but the left anterior descending coronary artery was not ligated. Normal saline was administered by gavage at a rate of 0.1 mL / kg. (2) Myocardial infarction model group (MI, n=20): Open chest surgery and ligation of the left anterior descending coronary artery were performed, and no drugs were administered by gavage after the operation; (3) Low-dose 3-HPP treatment group (L-3-HPPA, n=20): The drug was administered at a low dose of 30 mg / kg / d, with a drug concentration of 3 mg / mL and an oral gavage volume of 0.1 mL / kg; (4) High-dose 3-HPPA treatment group (H-3HPPA, n=20): The drug was administered at a high dose of 90 mg / kg / d, with a drug concentration of 9 mg / ml and an oral gavage volume of 0.1 mL / kg; (5) Coronary Heart Disease Formula No. 1 Treatment Group (ZY, n=20): The drug concentration was 11.05 g / kg, and the oral administration volume was 0.1 mL / kg; (6) Captopril positive control group (KT, n=20): The dosage was 10 mg / kg and the gavage volume was 0.1 mL / kg. In addition, all groups maintained a regular diet and water intake.

[0040] 2. Echocardiography of mice After drug administration, cardiac function in mice was assessed using a Vevo3100 ultra-high resolution small animal ultrasound imaging system. Mice were first anesthetized with isoflurane, and hair was removed from the left chest. The mice were then fixed to a control table, and a green transparent gel conductor was applied. The heart rate was maintained at 400-500 bpm using the ultrasound imaging system. A high-frequency probe was used to locate the mouse heart, probing at the mitral and horizontal positions. Short-axis views were taken parasternally for B-mode and M-mode measurements. Based on the experimental animals and objectives, this experiment primarily measured left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS), and echocardiograms were recorded.

[0041] 3. Measurement of organ coefficients in mice After completing the cardiac function test, the mice were weighed and their body weight (BW) recorded. After anesthetizing the mice with tribromoethanol, blood was collected from their eyeballs. The mice were then euthanized by cervical dislocation for tissue sampling. The thoracic cavity of the mice was quickly opened, the heart was removed, and the blood was rinsed with pre-cooled saline. After absorbing surface moisture with absorbent paper, the organ weight was quickly measured and the heart weight (HW) recorded. The HW / BW ratio was calculated. The left leg of the mouse was cut off, and the muscles and excess tissue on the surface of the tibia were dissected. The tibia length (TL) was measured with calipers, and the HW / TL ratio was calculated.

[0042] 4. ELISA was used to measure the levels of serum inflammatory factors IL-6 and IL-1β in mice. After blood collection from the eyeballs, the samples were left to stand on ice for 60-90 minutes, pre-cooled at 4°C, and centrifuged at 3000 rpm for 10 minutes. The supernatant was then collected as serum samples. Interleukin-6 (IL-6) and interleukin-1β (IL-1β) in mouse serum were detected using a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) kit.

[0043] First, equilibrate the aluminum foil bag in the kit to room temperature for 20 minutes. Then, remove the required strips. Unused strips should be sealed in a resealable bag and stored at 4°C. Set up standard wells and sample detection wells, adding 50 μL of sample to each well. Add 50 μL of different concentrations of standard to the standard wells. Dilute serum samples with diluent at a ratio of 1:4 according to the preliminary experimental results. Do not add any sample to the blank wells.

[0044] Subsequently, 100 μL of horseradish peroxidase (HRP)-labeled antibody was added to the standard wells and sample wells, respectively, while the blank wells were left untreated. The reaction wells were sealed with a sealing film and incubated at 37°C for 60 min. The liquid in the wells was then discarded and patted dry on absorbent paper. Each well was then filled with 350 μL of washing buffer, allowed to stand for 1 minute, and then discarded. This washing step was repeated 5 times.

[0045] Next, add 50 μL of substrate A and B to each well, incubate in a 37°C incubator for 15 min, add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0046] Based on the OD values ​​and concentrations of the standards, a standard curve is plotted, and the OD values ​​of the samples are substituted into the linear regression equation to calculate the corresponding concentrations of the samples.

[0047] The standard concentrations in the IL-6 kit are 120, 60, 30, 15, 7.5, and 3.75 pg / mL, respectively; the concentrations in the IL-1β kit are 120, 60, 30, 15, 7.5, and 3.75 pg / mL, respectively.

[0048] 5. TTC staining (1) 1% TTC working solution: Weigh 0.3 g TTC powder and dilute to 30 mL with PBS buffer (pH=7.4).

[0049] (2) After the fresh heart tissue is quick-frozen with dry ice, it is cut into tissue slices 3-5 mm thick. Each heart is cut into 4-5 slices and placed in a 24-well plate.

[0050] (3) Immerse the slices in 1% TTC solution and incubate at 37°C in a shaker in the dark for 15-30 minutes. Observe carefully to prevent over-staining.

[0051] (4) After incubation, transfer the slices to a 10% formaldehyde solution and fix them overnight in the dark. Then take them out, blot the formaldehyde liquid with filter paper, lay the slices flat, and take pictures.

[0052] 6. qPCR detection of relative expression levels of cardiac mRNA (1) Extraction of RNA from cardiac tissue Approximately 10 mg of left ventricular tissue was excised from mouse hearts on ice, and RNA was extracted from the heart tissue using a rapid tissue / cell extraction kit. First, the excised heart tissue and pre-chilled grinding beads were placed in EP tubes for grinding. 500 μL of lysis buffer was added to each tube, and the tissue was ground using a cryo-tissue homogenizer. Lysis was performed on ice for 20 min to ensure complete lysis. The tissue was centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was transferred to a gDNA removal column, centrifuged at 12000 rpm for 1 min, and the filtrate was collected. 0.5 volumes of anhydrous ethanol were added to the filtrate, and the mixture was thoroughly mixed using a pipette. The entire mixture was transferred to an RNase-free adsorption column, centrifuged at 13000 rpm for 30 s, and the filtrate was discarded. 700 μL of protein removal buffer was added to the adsorption column, centrifuged at 13000 rpm for 30 s, and the filtrate was discarded. Add 500 μL of wash buffer to the adsorption column, centrifuge at 13000 rpm for 30 s, discard the filtrate, and repeat once. Centrifuge an empty tube at 13000 rpm for 2 min. Remove the adsorption column and air dry for 10 min. Add 30 μL of RNase-free ddH2O to the center of the adsorption column, incubate at room temperature for 2 min, and centrifuge at 13000 rpm for 1 min to obtain the RNA sample. The RNA sample concentration was then determined using Nanodrop.

[0053] (2) RNA is reverse transcribed into cDNA RNA was reverse transcribed into cDNA using the AG RT Master Mix for with qPCR with gDNA Remover kit, and the mixture was prepared on ice according to the proportions described in Table 1.

[0054] Table 1. Reverse transcription reaction system (20 μL)

[0055] Set the reaction program on the PCR instrument: incubate at 37°C for 2 min, incubate at 55°C for 15 min, heat at 85°C for 5 min, and the resulting cDNA product can be diluted 5 times before use in qPCR experiments.

[0056] (3) Quantitative real-time PCR (qPCR) Experiments were performed using the Bright Cycle Universal SYBR Green qPCR Mix with UDG kit, with mouse GAPDH or ACTB as internal controls. The reaction system is shown in Table 2, and the primer sequences used are shown in Table 3. The quantitative PCR reaction program was set according to the kit: incubation at 37°C for 2 min, pre-denaturation at 95°C for 3 min, annealing at 95°C for 5 s, extension at 60°C for 34 s, for 40 cycles. Melting curve collection was performed using the default settings. The relative expression levels of each gene were calculated using the 2-ΔΔCT method.

[0057] Table 2 qPCR reaction system (20 μL)

[0058] Table 3 Primer Sequences

[0059] Statistical methods: All experimental results are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis and graphing were performed using GraphPadPrism 9.0 software. One-way ANOVA was used for comparisons among multiple groups, and t-test was used for comparisons between two groups. p < 0.05 was considered statistically significant.

[0060] II. Experimental Results 1. 3-HPPA can improve cardiac function in mice with LAD-induced myocardial infarction. like Figure 1 As shown, the effect of 3-HPPA on cardiac function in MI mice was evaluated using small animal ultrasound after four weeks of treatment. The results showed that compared with the sham-operated group (CON), the ejection fraction (EF) of the MI model mice decreased from 66% to 48%, and the fractional shortening (FS) decreased from 37% to 24%, indicating significantly impaired cardiac function. After four weeks of intervention with low-dose 3-HPPA (L group), EF recovered to 64% (a 16% increase compared to the MI group), and FS recovered to 29% (a 5% increase compared to the MI group). After four weeks of intervention with high-dose 3-HPPA (H group), EF recovered to 69% (a 21% increase compared to the MI group), and FS recovered to 33% (a 9% increase compared to the MI group). Overall, both low- and high-dose 3-HPPA improved cardiac function in MI mice to varying degrees. The high-dose 3-HPPA showed a greater improvement in both EF and FS than the low-dose group, and the EF in the high-dose group slightly exceeded that in the CON group.

[0061] 2,3-HPPA can alleviate myocardial hypertrophy and improve the survival rate of mice. Heart-to-weight ratio and heart-to-tibia ratio are indicators of cardiac health in animals. A high ratio may indicate cardiac hypertrophy and can be used to help assess the severity of myocardial infarction. For example... Figure 2 As shown, the heart-to-weight ratio decreased significantly in all 3-HPPA treatment groups, while the heart-to-tibia ratio did not change significantly, suggesting that myocardial infarction in mice may not have been accompanied by myocardial hypertrophy; the mortality rate of mice after MI surgery was as follows: Figure 2 As shown, deaths mainly occurred within two weeks after surgery. The mortality rate was high in the MI group, with a survival rate of only 75% for mice. In contrast, the survival rate was improved in all treatment groups, with a survival rate of 90% for mice in the low-dose 3-HPPA (L group) and 80% for mice in the high-dose 3-HPPA (H group).

[0062] 3. 3-HPPA can significantly reduce the levels of inflammatory factors IL-6 and IL-1β in mice with myocardial infarction. IL-6 (interleukin-6) and IL-1β (interleukin-1β) are key inflammatory factors. Their levels rise rapidly after myocardial infarction. Measuring changes in their levels helps assess the degree of inflammatory response and myocardial damage after myocardial infarction, and is beneficial for evaluating the effectiveness of drug treatment. Figure 3 As shown, compared with the sham group, the serum IL-6 level in the myocardial infarction model group (MI) mice increased from 52 pg / mL to 145 pg / mL (an increase of 178.8%), and the IL-1β level increased from 108 pg / mL to 138 pg / mL (an increase of 27.8%), suggesting that myocardial infarction induces a significant inflammatory response and myocardial damage. After 3-HPPA intervention, the levels of inflammatory factors in each treatment group decreased to varying degrees. Among them, low-dose 3-HPPA (L group) reduced IL-6 level to 120 pg / mL (17.2% lower than MI group) and IL-1β level to 132 pg / mL (4.3% lower than MI group); high-dose 3-HPPA (H group) reduced IL-6 level to 85 pg / mL (41.4% lower than MI group) and IL-1β level to 120 pg / mL (13.0% lower than MI group), with significantly better anti-inflammatory effect than the low-dose group; other control intervention groups (ZY, KT) also reduced the levels of inflammatory factors to varying degrees, among which the KT group had the strongest inhibitory effect on IL-6 (reduction of 48.3%), and the inhibitory effect on IL-1β was similar to that of the high-dose 3-HPPA group.

[0063] 4. 3-HPPA can significantly reduce the infarct area in mice with myocardial infarction. TTC staining results show that the infarcted area appears pale white, while normal myocardium appears red, which can visually reflect the size of the infarct area. Figure 4 As shown, compared with the sham group, the proportion of infarcted area in the heart of mice in the myocardial infarction model group (MI) was significantly increased, accounting for approximately 40%–50% of the heart cross-section, indicating that the myocardial infarction model was successfully established. After 3-HPPA intervention, the infarct area in each treatment group decreased to varying degrees. Low-dose 3-HPPA (L group) reduced the proportion of infarcted area to 10%–15%, a reduction of approximately 62.5%–75% compared to the MI group; high-dose 3-HPPA (H group) further reduced the proportion of infarcted area to 5%–10%, a reduction of approximately 75%–87.5% compared to the MI group, with significantly better infarct improvement than the low-dose group; other control intervention groups (ZY, KT) also effectively reduced the infarct area, with the proportion of infarcted area decreasing to 15%–20% and 20%–25%, respectively, both significantly lower than the MI group. Overall, 3-HPPA can dose-dependently reduce the myocardial infarction area in MI mice, with the protective effect of high-dose 3-HPPA being more prominent. 5. 3-HPPA can significantly reduce the mRNA expression levels of myocardial injury-related genes ANP, BNP, and MYH7. ANP and BNP are marker genes for myocardial injury, and MYH7 (β-myosin heavy chain) is an important component of myosin in cardiomyocytes, reflecting the remodeling process of cardiomyocytes after infarction, such as hypertrophy and fibrosis. Upregulation of all three genes is closely related to decreased cardiac function. Figure 5As shown, compared with the sham surgery group, the levels of ANP, BNP and MYH7 in the MI group were significantly increased, with ANP increasing from 1.0 to 3.8 (an increase of 280%), BNP increasing from 1.0 to 2.4 (an increase of 140%), and MYH7 increasing from 1.0 to 1.55 (an increase of 55%), suggesting that MI induced significant myocardial injury and pathological remodeling. All treatment groups showed varying degrees of reduction in the expression levels of these indicators. Low-dose 3-HPPA (L group): ANP decreased to 2.8 (a 26.3% reduction compared to the MI group), BNP decreased to 1.2 (a 50.0% reduction), and MYH7 decreased to 0.58 (a 62.6% reduction). High-dose 3-HPPA (H group): ANP decreased to 1.9 (a 50.0% reduction), BNP decreased to 1.1 (a 54.2% reduction), and MYH7 decreased to 0.45 (a 71.0% reduction), with significantly better overall inhibitory effects than the low-dose group. Other control intervention groups (ZY, KT) also effectively reduced gene expression. These results indicate that high concentrations of 3-HPPA can significantly downregulate the expression of genes related to myocardial injury and remodeling in MI mice, effectively alleviating myocardial pathological remodeling. 6. 3-HPPA can significantly reduce the mRNA expression levels of inflammatory factors IL-6, IL-1β, and TNF-α. The degree of inflammatory response in a mouse model of myocardial infarction can be assessed by detecting changes in the levels of IL-1β, IL-6, and TNF-α. Figure 6As shown, compared with the sham group, the expression of the above-mentioned inflammatory factors in the cardiac tissue of myocardial infarction model mice was significantly upregulated: IL-1β increased from 1.0 to 1.8 (an increase of 80.0%), IL-6 increased from 1.0 to 3.6 (an increase of 260.0%), and TNF-α increased from 1.0 to 3.0 (an increase of 200.0%). After treatment with low and high concentrations of 3-HPPA, traditional Chinese medicine compound, and captopril, the expression levels were significantly reduced. Low-dose 3-HPPA (L group) reduced IL-1β to 1.3 (a decrease of 27.8%), IL-6 to 2.0 (a decrease of 44.4%), and TNF-α to 1.8 (a decrease of 40.0%), initially inhibiting the progression of inflammation. High-dose 3-HPPA (H group) showed a significantly enhanced anti-inflammatory effect, reducing IL-1β to 1.2 (a decrease of 33.3%) and IL-6 to... The levels of IL-6 and TNF-α decreased to 1.6 (a 55.6% reduction) and TNF-α decreased to 1.2 (a 60.0% reduction), with particularly prominent inhibitory effects on IL-6 and TNF-α, showing significantly better anti-inflammatory effects than the low-dose group. The traditional Chinese medicine compound (ZY group) and captopril (KT group) also exhibited good anti-inflammatory activity. The ZY group showed the best inhibitory effect on IL-6 (a 58.3% reduction), while the KT group effectively reduced TNF-α expression (a 66.7% reduction). 7. 3-HPPA can improve the mRNA expression levels of apoptosis factors Bax, Bcl2, and Caspase-3 in mice with myocardial infarction. Bax, Bcl2, and Caspase-3 are key factors related to apoptosis, and an imbalance in their expression is a core molecular mechanism of cardiomyocyte apoptosis after myocardial infarction. Figure 7As shown, compared with the sham-operated group, the model group showed a significant increase in the mRNA levels of pro-apoptotic factors Bax and Caspase-3, and a significant decrease in the level of anti-apoptotic factor Bcl2. Specifically, Bax increased from 1.0 to 2.3 (an increase of 130%), Caspase-3 increased from 1.0 to 4.0 (an increase of 300%), and Bcl2 decreased from 1.0 to 0.55 (a decrease of 45%). After drug treatment, low-dose 3-HPPA (L group) reduced Bax to 2.1 (a decrease of 8.7%) and Caspase-3 to 2.5 (a decrease of 37.5%), while restoring Bcl2 to 0.7 (an increase of 27.3%), mildly inhibiting cardiomyocyte apoptosis. High-dose 3-HPPA (H group) showed a significantly enhanced anti-apoptotic effect, reducing Bax to 1.3 (a decrease of 43.5%) and Caspase-3 to 0.55 (a decrease of 45%). The concentration of Bcl-3 decreased to 2.0 (a 50.0% reduction) and increased Bcl-2 to 0.72 (a 30.9% increase), showing significantly better inhibitory effects on pro-apoptotic factors than the low-dose group. The traditional Chinese medicine compound (ZY group) and captopril (KT group) also exhibited good anti-apoptotic activity, with the KT group showing the strongest inhibitory effect on Caspase-3 (a 62.5% reduction). 8. 3-HPPA can improve the expression levels of NF-κB and Collagen I (type I collagen) in mice with myocardial infarction. NF-κB is a core transcription factor regulating immune and inflammatory responses; its overactivation exacerbates myocardial damage and subsequent fibrosis. Collagen I is an important indicator for assessing myocardial fibrosis, and its expression level directly reflects the degree of myocardial interstitial fibrosis. Figure 8As shown, compared with the sham-operated group, the mRNA expression of NF-κB and Collagen I in the myocardial tissue of mice in the myocardial infarction model group (MI) was significantly upregulated: NF-κB increased from 1.0 to 2.5 (an increase of 150%), and Collagen I increased from 1.0 to 1.9 (an increase of 90%), suggesting that the MI model successfully induced inflammatory activation and myocardial fibrosis. After 3-HPPA intervention, the abnormal expression of the above indicators was effectively reversed. Low-dose 3-HPPA (L group) reduced NF-κB to 1.7 (32% reduction) and Collagen I to 1.7 (10.5% reduction), mildly inhibiting the process of inflammation and fibrosis. High-dose 3-HPPA (H group) intervention showed significantly enhanced effect, reducing NF-κB to 1.5 (40% reduction) and Collagen I to 1.3 (31.6% reduction), with significantly better inhibitory effect on inflammation and fibrosis than the low-dose group. The traditional Chinese medicine compound (ZY group) and captopril (KT group) also showed good intervention activity. The ZY group showed more prominent inhibitory effects on NF-κB and Collagen I, while the KT group had a similar effect to the high-dose 3-HPPA group.

Claims

1. 3-HPPA in the preparation of drugs for the prevention and / or treatment of myocardial infarction. The structural formula of the 3-HPPA is shown in formula (I): 。 2. The application according to claim 1, characterized in that: The myocardial infarction includes ST-segment elevation myocardial infarction.

3. The application according to claim 1, characterized in that: The 3-HPPA comprises a pharmaceutically acceptable salt.

4. The application according to claim 1, characterized in that: The drug includes pharmaceutically acceptable excipients.

5. The application according to claim 1, characterized in that: The dosage forms of the drug include tablets, capsules, granules, injections, oral liquids, or sustained-release formulations.

6. The application according to claim 1, characterized in that: The drug is administered to mammals; Preferably, the mammal includes humans.

7. The application according to claim 1, characterized in that: The drug also includes any one or more other active ingredients; The other active ingredients have the function of treating myocardial infarction.

8. The application according to any one of claims 1 to 7, characterized in that: The drug has one or more of the following effects: 1) Improves cardiac contractile function after myocardial infarction; 2) Reduce the area of ​​myocardial infarction; 3) Inhibits the gene expression level of BNP in myocardial tissue; 4) Reduce the expression levels of inflammatory factors IL-6 and TNF-α in myocardial tissue; 5) Inhibit the expression levels of myocardial fibrosis-related factors TGF-β and Collagen I; 6) It exerts a cardioprotective effect by inhibiting the Wnt2 / β-catenin signaling pathway.

9. A pharmaceutical composition comprising a therapeutically effective amount of 3-HPPA or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 9, characterized in that: The pharmaceutical composition further includes any one or more other active ingredients; The other active ingredients have the function of treating myocardial infarction.

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