Application of small molecule compound E229-0305 in preparation of medicine for preventing or treating myocardial fibrosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly provides a small molecule compound E229-0305 for preparing a medicine for treating myocardial fibrosis. The small molecule compound E229-0305 can target PTGS2 protein, inhibit fibrosis of myocardial fibroblasts induced by TGF-beta1, reduce proliferation and migration capabilities of the myocardial fibroblasts, inhibit the fibroblasts from being converted into myofibroblasts, and inhibit expression of fibrosis-related genes and proteins. Besides, in a model of myocardial fibrosis after myocardial infarction of a mouse, the small molecule compound E229-0305 improves the cardiac function of the mouse with myocardial infarction and reduces the fibrosis area and collagen deposition of the heart. The small molecule compound E229-0305 can be used as a brand-new targeted PTGS2 small molecule inhibitor for preparing medicines for preventing and treating myocardial fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of the small molecule compound E229-0305 in preparing a drug for preventing or treating myocardial fibrosis. Background Art

[0002] Myocardial fibrosis is characterized by abnormal fibroblast proliferation, fibroblast-to-myofibroblast transformation, and excessive extracellular matrix deposition. It is a common finding in a variety of cardiovascular diseases, including myocardial infarction, hypertensive heart disease, ischemic cardiomyopathy, and diabetic cardiomyopathy. Under various stresses, such as myocardial injury, resident myocardial fibroblasts activate and transform into myofibroblasts to maintain the structural and functional integrity of the damaged heart. Excessive fibrosis can lead to systolic and diastolic dysfunction, arrhythmias, and even increase the risk of sudden cardiac death. Molecular pathways that contribute to myocardial fibrosis include the renin-angiotensin-aldosterone system (RAAS), transforming growth factor-β (TGF-β), oxidative stress, and inflammatory responses. Despite advances in therapeutic intervention, the underlying mechanisms regulating cardiac fibrosis remain unclear, necessitating the exploration of novel molecular targets to improve clinical outcomes.

[0003] PTGS2 / COX-2 (cyclooxygenase-2) is an enzyme that converts arachidonic acid into prostaglandins, which can enhance tumor progression by inhibiting apoptosis and promoting proliferation and angiogenesis. Furthermore, the PTGS2 gene is associated with the risk of myocardial infarction (MI) and stroke, and PTGS2 plays an important role in MI treatment. PTGS2 has low basal expression levels in most tissues and cell types, but is highly expressed in response to numerous stimuli, such as cytokines, growth factors, and xenobiotics. PTGS2 production is associated with tissue fibrosis. Previous studies have shown that PTGS2 protein expression is elevated in infarcted myocardial tissue, and the PTGS2 inhibitor NS-398 can inhibit PTGS2 protein expression and reduce collagen deposition in the heart after myocardial infarction. Therefore, exploring new small molecule compounds targeting PTGS2 is of great significance for the clinical drug development of fibrosis-related diseases.

[0004] Small molecule compounds hold great potential for the treatment of cardiac fibrosis. They can act through multiple mechanisms, including modulating cell signaling pathways, improving cellular metabolism, and inhibiting the fibrotic process. Published studies have shown that small molecule compounds can inhibit cardiac fibrosis by targeting specific cell signaling pathways, such as TGF-β / Smad and Wnt / β-catenin, thereby reducing the expression of fibrosis-related genes. Furthermore, some small molecule compounds possess antioxidant properties, capable of scavenging harmful reactive oxygen species, mitigating oxidative stress damage to heart cells, and protecting cardiac function.

[0005] The small molecule compound E229-0305 is an organic compound containing multiple benzene rings, pyrrole rings, and piperazine rings. Its Chinese name is 1-(2,3-dimethylphenyl)-4-(1-{pyrrole, 2-A]quinoxaline-4-yl}piperidine-3-carbonyl)piperazine, and its English name is 1-(2,3-DIMETHYLPHENYL)-4-(1-{PYRROLO[1,2-A]QUINOXALIN-4-YL}PIPERIDINE-3-CARBONYL)PIPERAZINE. Its molecular formula is C 26 H 33 N5O, chemical formula is There are no reports on the role of the small molecule compound E229-0305 in myocardial fibrosis. Summary of the Invention

[0006] In view of the problems of myocardial fibrosis being easy to develop and being harmful, the present invention aims to provide the use of the small molecule compound E229-0305 in the preparation of a drug for preventing and treating myocardial fibrosis, wherein the small molecule compound E229-0305, PubChem number: 328610468, relative molecular mass: 467.3 g / mol, chemical structure:

[0007] The present invention uses TGF-β1 and myocardial infarction models to construct in vitro and in vivo myocardial fibrosis models, respectively. After treatment with compound E229-0305, fibrosis-related mRNA and protein indicators are detected. It is found that compound E229-0305 can significantly reduce the protein and mRNA levels of α-smooth muscle actin (α-SMA), type I collagen (COL-1), and fibronectin (FN1) in cardiac fibroblasts and infarcted myocardial tissue. In addition, compound E229-0305 can improve cardiac function in mice with myocardial infarction and reduce the area of ​​fibrosis and collagen deposition. The present invention confirms that the small molecule compound E229-0305, as a new drug, can be used to prevent and treat myocardial fibrosis and has good clinical application value.

[0008] This application adopts the following technical solutions:

[0009] In a first aspect, the present invention provides the use of a small molecule compound E229-0305 in the preparation of a drug for preventing or treating myocardial fibrosis. The structural formula of the small molecule compound E229-0305 is:

[0010]

[0011] In the above technical solution, the small molecule compound E229-0305 inhibits TGF-β1-induced cardiac fibroblast proliferation, migration and transformation of fibroblasts into myofibroblasts.

[0012] In the above technical solution, the small molecule compound E229-0305 inhibits the high expression of α-SMA, COL-1, FN1 and PTGS2 proteins and mRNA in cardiac fibroblasts induced by TGF-β1.

[0013] In the above technical solution, the small molecule compound E229-0305 inhibits the expression of α-SMA, COL-1, FN1 and PTGS2 proteins and mRNA in the heart tissue of mice with myocardial infarction.

[0014] In the above technical solution, the drug uses the small molecule compound E229-0305 as the active ingredient, and its concentration is 1 nM to 1 mM, and more preferably 5 to 20 μM.

[0015] In the above technical solution, the drug reduces the transcription level and protein expression level of α-SMA, COL-1 and FN1 through the small molecule compound E229-0305, thereby exerting the ability to prevent or treat myocardial fibrosis.

[0016] In the above technical solution, the drug is composed of a compound E229-0305 at an effective dose, which can be a single component or a composition containing an effective dose of the compound E229-0305. The drug can include pharmaceutically acceptable excipients.

[0017] In the above technical solutions, the dosage form of the drug is injection, tablet, powder injection, granule, capsule, oral solution, pill, gel, suppository, ointment, emulsion, mixture, suspension or cream.

[0018] In the above technical solution, the excipient is one or more of a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, a flavoring or other pharmaceutically acceptable excipients.

[0019] In the above technical solutions, the administration routes of the drug include oral administration, injection, respiratory inhalation, topical administration, sublingual administration and rectal administration.

[0020] In the above technical solution, the myocardial fibrosis includes myocardial fibrosis caused by myocardial infarction, diabetes, hypertension, myocarditis, valvular heart disease, heart failure, coronary heart disease, etc.

[0021] In a second aspect, the present invention provides a pharmaceutical composition comprising an effective dose of the small molecule compound E229-0305 as an active ingredient, wherein the structural formula of the small molecule compound E229-0305 is: Its concentration is 1nM~1mM.

[0022] The present invention has the following beneficial effects:

[0023] Compared to existing myocardial fibrosis treatments, the small molecule compound E229-0305 offers a novel therapeutic agent for treating the development and progression of myocardial fibrosis. It exhibits significant therapeutic efficacy against myocardial fibrosis, inhibiting the transcription and protein expression of α-SMA, COL-1, and FN1, potentially preventing or treating myocardial fibrosis. In a mouse model of myocardial fibrosis established by ligation of the left anterior descending coronary artery, intraperitoneal injection of 10 mg / kg of the small molecule compound E229-0305 significantly improved the ejection fraction and fractional shortening of the mice with myocardial fibrosis, inhibited the transcription and translation of fibrosis-related genes, and alleviated myocardial fibrosis. The use of the small molecule compound E229-0305 as an active ingredient in the preparation of a drug with the potential to alleviate myocardial fibrosis offers new insights into the clinical treatment and prevention of myocardial fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effects of the small molecule compound E229-0305 in Example 1 on the proliferation and migration of primary neonatal rat cardiac fibroblasts induced by TGF-β1 are shown. A shows the effect of different concentrations of compound E229-0305 on cardiac fibroblast viability after TGF-β1 induction using a CCK8 assay. B and C show the effect of compound E229-0305 on cardiac fibroblast migration using a wound healing assay. D and E show the effect of compound E229-0305 on the expression of the proliferation marker Ki67 in cardiac fibroblasts using immunofluorescence. Data were analyzed using one-way analysis of variance. * P<0.05, ** P<0.01, compared with the control group. # P<0.05, ## P<0.01, compared with TGF-β1 group.

[0025] Figure 2The effect of the small molecule compound E229-0305 on the mRNA and protein expression of fibrosis markers α-SMA, COL-1, and FN1 in TGF-β1-induced primary rat cardiac fibroblasts. A, B, and C show the mRNA expression of α-SMA, COL-1, and FN1 in Example 2, respectively. D, E, F, and G show the effect of compound E229-0305 on the protein expression of α-SMA, COL-1, and FN1 in Example 2. H and I show the effect of TGF-β1 and compound E229-0305 on the fluorescence intensity of α-SMA, a marker for the transformation of primary rat cardiac fibroblasts into myofibroblasts, in Example 3. Data were analyzed using one-way analysis of variance. * P<0.05, ** P<0.01, compared with the control group. ## P<0.01, compared with TGF-β1 group.

[0026] Figure 3 Figure 4 shows target screening for the treatment of myocardial fibrosis with the small molecule compound E229-0305 and interaction analysis between compound E229-0305 and the PTGS2 protein. A is a Venn diagram of the intersection of predicted targets for myocardial fibrosis disease processes in the Disgenet, Genecards, Ncbi, and Omim databases. B is a Venn diagram of the intersection of predicted targets for the small molecule compound E229-0305 in the Swiss, Charite, Sea, and Calcnet databases. C is a Venn diagram of the intersection of predicted targets for myocardial fibrosis and predicted targets for the small molecule compound E229-0305. D is a PPI protein interaction analysis of the predicted targets of the small molecule compound E229-0305. E is a PPI protein interaction analysis of the intersection of targets for compound E229-0305 and targets for myocardial fibrosis. F is the molecular docking results of the small molecule compound E229-0305 and the PTGS2 protein. G is a thermal shift experiment to evaluate the effect of small molecule compound E229-0305 on the thermal stability of PTGS2 protein. H and I are the effects of small molecule compound E229-0305 on PTGS2 protein expression. The data were analyzed using one-way analysis of variance. ** P<0.01.

[0027] Figure 4The effect of the small molecule compound E229-0305 in Example 5 on the cardiac function of mice after myocardial infarction injury. A is a schematic diagram of the construction of the myocardial infarction model and the administration process of compound E229-0305. B is the results of cardiac ultrasound of mice in the sham operation group, myocardial infarction model group and the group treated with small molecule compound E229-0305 after myocardial infarction surgery. C is the results of cardiac ejection fraction of the three groups of mice; D is the results of cardiac short axis shortening rate of the three groups of mice; E is the results of left ventricular end-diastolic diameter of the three groups of mice; F is the left ventricular end-diastolic volume of the three groups of mice. The data were statistically analyzed using one-way analysis of variance. * P<0.05, ** P<0.01.

[0028] Figure 5 These are the results of HE staining, Masson staining, and Sirius red staining in Example 5.

[0029] Figure 6 This figure shows the effect of the small molecule compound E229-0305 in Example 5 on the mRNA expression of α-SMA, COL-1, FN1, and PTGS2 in cardiac tissue. A, B, C, and D show the mRNA expression of α-SMA, COL-1, FN1, and PTGS2 in cardiac tissue in the sham-operated group, the MI model group, and the MI model plus compound E229-0305 group, respectively. Data were analyzed using one-way analysis of variance. * P<0.05, ** P<0.01.

[0030] Figure 7 This figure shows the effect of the small molecule compound E229-0305 in Example 5 on the expression of α-SMA, COL-1, FN1, and PTGS2 proteins in cardiac tissue. A, B, C, D, and E show the expression of α-SMA, COL-1, FN1, and PTGS2 proteins in cardiac tissue in the sham surgery group, the MI model group, and the MI model plus compound E229-0305 group. Data were analyzed using one-way analysis of variance. * P<0.05, ** P<0.01. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1: Small molecule compound E229-0305 inhibits TGF-β1-induced proliferation and migration of cardiac fibroblasts

[0033] Heart tissue was extracted from 1-3 day old C57BL / 6 neonatal mice and primary myocardial fibroblasts were isolated and cultured. The experiment was first divided into a control group, a TGF-β1-induced group, and a TGF-β1-plus-four different concentrations of the small molecule compound E229-0305 group. The cell viability of myocardial fibroblasts in different groups was detected by CCK8 assay. The results showed that TGF-β1 can promote the proliferation of myocardial fibroblasts and increase cell viability, while the addition of the small molecule compound E229-0305 can inhibit the excessive proliferation of myocardial fibroblasts and reduce cell viability. At 20μM, cell viability was significantly reduced compared with the TGF-β1 group ( Figure 1 A), so the subsequent experiments selected three different concentrations of 5μM, 10μM and 20μM as treatment groups. The migration and proliferation abilities of cardiac fibroblasts were then tested by wound healing assay and Ki67 immunofluorescence assay, respectively. The results showed that the small molecule compound E229-0305 effectively alleviated the migration of cardiac fibroblasts induced by TGF-β1 ( Figure 1 BC) and proliferation ( Figure 1 DE) ability.

[0034] The specific method is as follows:

[0035] 1. Extraction of primary myocardial fibroblasts from neonatal rats:

[0036] Select 1-3 day old C57BL / 6 mice, disinfect them by immersing them in 75% alcohol, then remove the hearts and place them in a dish containing pre-chilled PBS and a small amount of tertiary antibody. Transfer all hearts to a 50 mL centrifuge tube and wash twice with dhanks solution. For every 30 mice, add 3 mL of 0.25% trypsin and 2 mL of dhanks solution to each 50 mL centrifuge tube, seal the tube, and incubate at 4°C on a shaker overnight (12-14 hours).

[0037] The next day, discard the trypsin and dhanks. Rinse once with DMEM medium, then add type II collagenase solution and incubate at 37°C on a shaker at 160 rpm for 10 minutes to digest the heart. Add an equal volume of DMEM complete medium containing 10% FBS to the digestion supernatant. Repeat this step 2-3 times until the heart tissue is completely digested. Centrifuge the digested medium at 1000 rpm for 5 minutes, then discard the supernatant. Resuspend the pellet in DMEM complete medium containing 10% FBS, transfer the pellet to a culture flask, and incubate at a differential rate at 37°C for 1.5 hours. After 1.5 hours, remove the flask and use a Pasteur pipette to remove any adherent cardiomyocytes. The remaining adherent cells are primary neonatal rat cardiac fibroblasts. Add complete medium and continue incubation for 48 hours. Subsequent experiments can be performed when the cardiac fibroblast density reaches 90%.

[0038] 2. CCK8 assay to determine cell viability

[0039] Primary myocardial fibroblasts of neonatal rats in the logarithmic growth phase were collected and counted using a cell counting plate. The cell density was adjusted to 6*10 5 / mL, 100 μL of cell suspension was added to each well of a 96-well plate, with 6 replicates for each group. After culturing for 36 hours, 20 ng / mL TGF-β1 was added, and different concentrations of the small molecule compound E229-0305 were added at the same time. After continuing to culture for 48 hours, according to the requirements of the CCK8 kit, under light-proof conditions, the CCK8 stock solution was diluted 10 times using DMEM base medium. After discarding the original culture medium of the 96-well plate, the plate was washed once with PBS, and the diluted CCK8 working solution was added. The 96-well plate was placed in a 37°C incubator and incubated for 3 hours, and the absorbance at 450 nm was measured using a microplate reader.

[0040] 3. Scratch assay to detect cell migration ability

[0041] Primary myocardial fibroblasts of neonatal rats in logarithmic growth phase were digested and cultured at 3*10 5 Cells were seeded with 500 μg / mL of TGF-β1 in 6-well plates. When the cell density reached 70%-80%, two scratches of uniform width were made in each well using a 10 μL pipette tip perpendicular to the bottom of the plate. After washing the scratched cells with PBS, 20 ng / mL TGF-β1 and different concentrations of the small molecule compound E229-0305 were added. The scratches at 0 h were immediately photographed under a microscope, and the same positions were photographed at 24 h and 48 h.

[0042] 4. Immunofluorescence assay to detect the fluorescence intensity of Ki67 protein

[0043] Take cells in logarithmic growth phase and use 1.5*10 5 Cells were seeded at 100 μg / mL in a 24-well plate and cultured for 24 hours. The cells were then starved with 1% FBS for 12 hours, then incubated with 20 ng / mL TGF-β1 and various concentrations of the small molecule compound E229-0305 for an additional 48 hours. After 48 hours, the cells were removed from the incubator for immunofluorescence staining. 500 μL of 4% paraformaldehyde was added to each well for fixation at room temperature for 20 minutes. The cells were washed three times with PBS for 5 minutes each. The membrane was permeabilized with 0.5% Triton X-100 for 30 minutes at room temperature. The cells were washed three times with PBS for 5 minutes each. 1% goat serum was added and the cells were blocked at 37°C for 1.5 hours. 200 μL of primary antibody was added and the cells were shaken overnight at 4°C. 200 μL of secondary antibody was added and the cells were incubated at room temperature for 1.5 hours. The cells were washed three times with PBS for 5 minutes each. 200 μL of DAPI was added to each well for 5 minutes at room temperature. The cells were washed three times with PBS for 5 minutes each. 200 μL of PBS was added to each well and the cells were photographed under a fluorescence microscope.

[0044] Example 2: Small molecule compound E229-0305 inhibits TGF-β1-induced extracellular matrix deposition in cardiac fibroblasts

[0045] Heart tissue was extracted from 1-3 day old C57BL / 6 neonatal mice, and primary myocardial fibroblasts were isolated and cultured. The experimental groups were divided into control group, TGF-β1 induced group, and low, medium, and high concentrations of the small molecule compound E229-0305 induced group. Real-time quantitative PCR and Western Blot experiments were used to detect the transcription and translation levels of fibrosis-related markers α-SMA, COL-1, and FN1. It was found that the small molecule compound E229-0305 could significantly reduce the mRNA expression of α-SMA, COL-1, and FN1 induced by TGF-β1 ( Figure 2 AC) and protein ( Figure 2 DG) expression level increased, reducing extracellular matrix deposition.

[0046] The specific method is as follows:

[0047] 1. Detection of α-SMA, COL-1, and FN1 mRNA levels by real-time quantitative PCR

[0048] a) mRNA extraction

[0049] In this example, RNA was extracted using TransGen Biotech's TransZol conventional RNA extraction kit according to the kit instructions. Cell culture and treatment with TGF-β1 and compound E229-0305 were performed in the same manner as above. After 48 hours of drug incubation, 1 mL of TransZol was added to each well of the six-well plate to lyse the cells. The cells were transferred to a 1.5 mL eppendorf tube, 200 μL of chloroform was added, and the tubes were vigorously shaken for 15 seconds and incubated at room temperature for 3 minutes. The eppendorf tubes were then placed in a balanced centrifuge and centrifuged at 4°C at 10,000 x g for 10 minutes. 500 μL of the top aqueous phase was aspirated, an equal volume of isopropanol was added, and the mixture was mixed by inversion and incubated at room temperature for 30 minutes. Centrifuge at 10,000 x g for 10 min at 4°C, discard the supernatant, add 1 mL of 75% ethanol to the pellet for washing, centrifuge at 7,500 x g for 5 min at 4°C, discard the supernatant, and air-dry the pellet. Dissolve it in 10 μL of RNA Dissolving Solution, incubate in a metal bath at 55-60°C for 10 min, and determine the RNA concentration by UV spectrophotometry.

[0050] b) mRNA reverse transcription

[0051] In this example, RNA reverse transcription was performed using the TransScript All-in-One First-Strand cDNA Synthesis Kit (One-Step Removal of gDNA) (for qPCR) from TransGen Biotech. cDNA synthesis was performed according to the recommended amounts in the instructions. The specific amounts of each reagent and RNA used are shown in Table 1.

[0052] Table 1

[0053] Element volume Total RNA / mRNA ≤1μg / ≤100ng 5×TransScript All-in-One SuperMix for qPCR 4 μL gDNA Remover 1 μL Enzyme-free water Variable Total volume 10 μL

[0054] c) Fluorescence real-time quantitative PCR

[0055] The primers used for real-time fluorescence quantitative PCR were designed by searching the mRNA sequences on the National Center for Biotechnology Information (NCBI) website. The primer sequences are shown in Table 2.

[0056] Table 2

[0057]

[0058]

[0059] In this example, fluorescent real-time quantitative PCR was performed using TransGen Biotech's Top dye method fluorescent quantitative PCR premix kit, and the reaction system is shown in Table 3.

[0060] Table 3

[0061]

[0062] The reaction system was prepared in eight PCR tube strips. After sample addition, all liquid was centrifuged to the bottom of the tube using a handheld centrifuge to avoid bubbles. The PCR program settings are shown in Table 4.

[0063] Table 4

[0064]

[0065] 2. Western Blot Detection of α-SMA, COL-1, and FN1 mRNA Content

[0066] a) Total cell protein extraction

[0067] Cell culture and treatment with TGF-β1 and compound E229-0305 were performed as described above. After 48 hours of incubation with the drugs, the old medium was discarded, and each well was washed once with 1 mL of PBS, which was then discarded. 35 μL of RIPA lysis buffer (strong) containing protease inhibitors was added to each well of the six-well plate. The cells were scraped with a cell scraper and transferred to a 1.5 mL EP tube. Sonication was performed three times, each for 10 seconds, with 5 minutes between cycles. The cells were centrifuged at 13,500 rpm for 15 minutes at 4°C. The supernatant, representing total cellular protein, was collected in a new 1.5 mL EP tube.

[0068] b) Protein concentration determination

[0069] In this example, protein concentration was determined using the GLPBIO BCA protein concentration assay kit. First, prepare the BCA working solution at a ratio of 50:1 (Solution A:Solution B). Add 2 μL of the protein stock solution, 18 μL of ultrapure water, and 160 μL of the BCA working solution to each well of a 96-well plate. Incubate at 37°C for 30 minutes using a microplate reader to measure absorbance at 562 nm. Protein concentration was calculated using a protein concentration standard. After aligning all samples to a uniform concentration, add 1 / 5 of the sample volume of 6× protein loading buffer, mix thoroughly, and denature by heating in a metal bath at 100°C for 10 minutes.

[0070] c) SDS-PAGE configuration

[0071] Wash the thin plate and 1.5 mm thick plate, then test for leaks with ultrapure water and blow dry. Prepare 10% separation gel according to the ingredients in Table 5, mix thoroughly, pour into the gel plate, and seal with ultrapure water.

[0072] Table 5

[0073] Element volume Ultrapure water 4.0mL 1.5M Tris-HCl, pH=8.8 2.5mL 30% acrylamide 3.3mL 10% SDS 0.1mL 10% ammonium persulfate 0.1mL Tetramethylethylenediamine 0.004mL Total volume 10mL

[0074] Once a clear line forms between the separating gel and the upper layer of ultrapure water, pour off the upper layer of ultrapure water and prepare a 5% concentrated gel with the ingredients shown in Table 6. Immediately after adding the concentrated gel, insert a comb of appropriate size to prevent air bubbles. After 40 minutes, remove the prepared gel from the gel rack and set aside.

[0075] Table 6

[0076]

[0077]

[0078] d) Electrophoresis

[0079] Prepare 1× electrophoresis buffer according to Table 7, place the glass plate in the electrophoresis tank, and remove the comb. Adjust the protein loading to 30 μg based on the BCA quantification results. Run the electrophoresis at a constant voltage of 70 V for 40 minutes, then switch to a constant voltage of 110 V for approximately 45 minutes. Stop the electrophoresis when the front end of the sample approaches the bottom of the separating gel.

[0080] Table 7

[0081] Element Dosage Tris(hydroxymethyl)aminomethane 2.72g Glycine 18.8g 10% SDS solution 10mL Ultrapure water 990mL Total volume 1000mL

[0082] e) Transfer

[0083] Prepare 1× transfer buffer according to Table 8 and pre-cool at 4°C. Cut a PVDF membrane to the appropriate size and activate it with methanol for 5 minutes. Remove the gel from the center of the glass plate and arrange it in the following order: black side of the transfer clip, sponge, filter paper, gel, PVDF membrane, filter paper, sponge, white side of the transfer clip. Clamp the transfer clip securely and place it into the transfer electrode core, ensuring the black side of the transfer clip faces the black plate of the core. Set the transfer current to a constant current of 300 mA and the transfer time to 90 minutes.

[0084] Table 8

[0085] Element Dosage Tris(hydroxymethyl)aminomethane 3.03g Glycine 14.4g Methanol 200mL Ultrapure water 800mL Total volume 1000mL

[0086] f) Closed

[0087] After the transfer was completed, the PVDF membrane was removed and directly placed in 5% skim milk powder for blocking at room temperature for 2 h.

[0088] g) Primary antibody incubation

[0089] After blocking, use PBS to wash away excess blocking solution, place the PVDF membrane and primary antibody solution in the antibody incubation box, and incubate on a shaker at 4°C for 14-16 hours.

[0090] h) Secondary antibody incubation

[0091] Recover the primary antibody and wash the PVDF membrane three times with PBST for 5 minutes each time. Add the secondary antibody solution corresponding to the primary antibody and incubate at room temperature for 1 hour.

[0092] i) Development

[0093] After secondary antibody recovery, the PVDF membrane was washed three times with PBST for 5 minutes each. The PVDF membrane was scanned upside down using the LICOR Odyssey dual-color infrared laser imaging system, and subsequent analysis was performed using Image Studio software.

[0094] Example 3: Small molecule compound E229-0305 inhibits TGF-β1-induced cardiac fibroblast to myofibroblast transformation

[0095] Heart tissue was extracted from 1-3 day old C57BL / 6 neonatal mice and primary myocardial fibroblasts were isolated and cultured. The experimental groups were: control group, TGF-β1 induced group, low, medium and high concentrations of small molecule compound E229-0305 induced group. Immunofluorescence experiment was used to fluorescently stain α-SMA protein, a marker of fibroblast to myofibroblast transformation, and its relative fluorescence intensity was analyzed. The results showed that under TGF-β1 induction, the fluorescence intensity of α-SMA protein increased significantly, and the transformation of fibroblasts to myofibroblasts increased. The addition of different concentrations of small molecule compound E229-0305 could reduce the fluorescence intensity of α-SMA to varying degrees, inhibiting the transformation of fibroblasts to myofibroblasts ( Figure 2 HI).

[0096] The specific method is as follows:

[0097] 1. Immunofluorescence assay to detect the fluorescence intensity of α-SMA protein

[0098] The immunofluorescence experiment method in this example is the same as that in Example 1.

[0099] Example 4: Target confirmation of the small molecule compound E229-0305 in inhibiting myocardial fibrosis

[0100] Through network pharmacology and bioinformatics methods, the small molecule compound E229-0305 and the downstream targets of myocardial fibrosis were screened from websites such as Disgenet, Genecrads, NCBI, Swiss and Sea that predict myocardial fibrosis and small molecule interacting proteins, and the intersection was obtained by Venn diagram ( Figure 3 AC), and screened out 6 proteins that may be downstream targets of the small molecule compound E229-0305 to inhibit myocardial fibrosis, namely: CTSD, PTGS2, HIF1A, MMP7, SLC6A4 and PARP1. Next, the possible downstream targets of the small molecule compound E229-0305 and the intersection of the small molecule compound E229-0305 and the downstream targets of myocardial fibrosis were analyzed using the STRING database for PPI protein interaction analysis, and it was found that PTGS2 interacted with many proteins ( Figure 3 DE). The molecular docking results of compound E229-0305 and PTGS2 protein showed ( Figure 3F), compound E229-0305 formed a hydrogen bond with the arginine at position 44 of the PTGS2 protein amino acid sequence, and the two had good binding affinity with a binding energy of -8.7 kcal / mol. Western Blot experiments showed that the expression of PTGS2 protein increased under TGF-β1 induction conditions, and after the addition of the small molecule compound E229-0305, the expression of PTGS2 protein decreased ( Figure 3 H). The above results demonstrate that compound E229-0305 can alleviate myocardial fibrosis by inhibiting PTGS2 protein expression.

[0101] The specific method is as follows:

[0102] 1. Small molecule compound E229-0305 and downstream target screening for myocardial fibrosis

[0103] The structural information of compound E229-0305 was collected using the Pubchem (pubchem.ncbi.nlm.nih.gov) database, and the chemical structure of each component was drawn using ChemDraw 3D software. The results were saved in sdf and mol file formats. The compound-related targets were predicted using platforms such as SwissTargetPrediction (www.swisstargetprediction.ch / ) and charite (https: / / prediction.charite.de / ). Furthermore, the prediction of myocardial fibrosis-related targets was specifically performed by using databases such as the OMIM database (www.omim.org), Genecards (www.genecards.org), and diagenet (www.disgenet.org / home / ) to enter the disease name "myocardial fibrosis" for retrieval and obtain myocardial fibrosis disease targets. The search results of each database were summarized and duplicate genes were removed to obtain myocardial fibrosis-related targets. Next, a Venn diagram was constructed for the key targets to obtain intersections. Specifically, the target information was imported into the Microbiological Information Platform (https: / / www.bioinformatics.com.cn / ) to draw a Venn diagram between compound E229-0305 and myocardial fibrosis-related targets to obtain drug-disease intersection targets.

[0104] The protein interaction (PPI) analysis was performed by importing the above-mentioned intersection targets into the STRING (https: / / string-db.org / ) database, selecting the species as "Mus musculus", setting the confidence data to a network relationship higher than 0.9, removing free proteins, and importing the results into Cytoscape 3.7.1 software to construct a PPI network diagram.

[0105] 2. Docking of the small molecule compound E229-0305 with the PTGS2 protein

[0106] The PDB database (https: / / www.rcsb.org / ) was used to search for target protein structures related to the core protein. A relatively high-resolution PTGS2 protein three-dimensional crystal structure (pdb format) file was selected. The corresponding pdb file was imported into Pymol software to remove water molecules, ligands, and other components. The file was then imported into AutoDockTools software for hydrogenation, charge calculation, and atomic rigidity determination. The structure was saved as a pdbqt file, which served as a protein receptor. The three-dimensional structure of the small molecule compound E229-0305 was drawn using Chemdraw 3D software and saved as a mol2 file. After full hydrogenation using AutoDockTools, the structure was further converted into a pdbqt file, yielding a small molecule ligand suitable for molecular docking.

[0107] Import the pdbqt format protein receptor and small molecule ligand files into AutoDockTools. Set the semi-flexible docking method to the box parameters and run AutoGrid to generate a gpf format file. Then run AutoDock to dock the receptor and small molecule ligand separately, generating a .dlg file. Analyze the .dlg file to determine the binding energy of the molecular docking. A binding energy of ≤ -1.2 kcal / mol is generally considered to indicate a stable binding between the receptor and ligand, and a reliable docking result. The docking results were then imported into Pymol software for visualization and analysis.

[0108] 3. Western Blot assay to detect PTGS2 protein content

[0109] The steps were consistent with those of the Western Blot experiment in Example 2.

[0110] 4. Cellular thermal shift experiment of small molecule compound E229-0305 and PTGS2 protein

[0111] Primary neonatal rat cardiac fibroblasts were collected and placed in 1.5 mL EP tubes. RIPA lysis buffer (strong) was added to resuspend the cells and sonicated three times for 10 seconds each, with 5 minutes between cycles. The cells were centrifuged at 13,500 rpm at 4°C for 15 minutes, and the supernatant was collected into a new 1.5 mL EP tube. The supernatant was divided equally into two aliquots. One aliquot was spiked with the small molecule compound E229-0305 at a final concentration of 50 μM, while the other aliquot was spiked with an equal amount of DMSO as a solvent control. After incubation at room temperature for 3 hours, the DMSO and E229-0305 groups were each aliquoted into nine 0.2 mL EP tubes, 50 μL per tube. The centrifuge tubes were heated at nine different temperatures (55, 60, 65, 70, 75, 80, 85, 90, and 95°C) for 25 min. After heating, the samples were placed on ice and centrifuged at 13,500 rpm for 15 min at 4°C. The supernatant was collected into a new 1.5 mL EP tube, and 1 / 5 volume of 6× protein loading buffer was added. The supernatant was denatured at 100°C for 10 min. The samples were stored at -80°C for subsequent Western blot analysis of PTGS2 and GAPDH protein levels.

[0112] Example 5: Small molecule compound E229-0305 protects cardiac function and slows myocardial fibrosis in mice after myocardial infarction

[0113] 6-8 week old male C57BL / 6 mice were selected and a post-myocardial infarction myocardial fibrosis model (MI) was established by ligating the left anterior descending coronary artery for 4 weeks. The experiment was divided into a sham operation group, an MI model group, and an MI model + compound E229-0305 treatment group. From the 2nd to the 26th day after myocardial infarction surgery, compound E229-0305 was administered by intraperitoneal injection every other day at a dose of 10 mg compound / kg body weight. On the 28th day, cardiac function of mice was tested using a small animal Doppler ultrasound imager. Compared with the sham operation group, the ejection fraction and short axis shortening rate of mice in the MI model group were significantly reduced, while the ejection fraction and short axis shortening rate of the compound E229-0305 treatment group were significantly improved compared with the MI model group ( Figure 4 The results of HE staining, Masson staining and Sirius red staining showed that compound E229-0305 significantly improved the inflammatory infiltration in the hearts of myocardial infarction mice and reduced the fibrosis area and collagen deposition ( Figure 5 By extracting proteins and RNA from the myocardial infarction area and performing real-time quantitative PCR and Western Blot experiments, it was found that the mRNA and protein expressions of fibrosis indicators α-SMA, COL-1, and FN1 in the heart tissue of myocardial infarction mice treated with compound E229-0305 were significantly reduced compared with the MI model group, and the transcription and translation levels of PTGS2 were also significantly reduced ( Figure 6 , Figure 7 The above results indicate that compound E229-0305 can improve cardiac function, reduce infarct size and collagen deposition, and alleviate myocardial fibrosis in mice with myocardial infarction-induced myocardial fibrosis by inhibiting the protein and mRNA expression of PTGS2 in the infarcted heart.

[0114] The specific method is as follows:

[0115] 1. Construction of a mouse model of myocardial fibrosis after myocardial infarction

[0116] Six- to eight-week-old male C57BL / 6 mice (weighing approximately 22 g) were selected and anesthetized with an intraperitoneal injection of Avertin (20 μL / g). After being fixed in a supine position on the operating table, the left chest was depilated and disinfected. An endotracheal tube was then inserted through the glottis and connected to a small animal ventilator with a respiratory rate of 110 bpm and a tidal volume of 0.2 mL. A 1.5 cm longitudinal incision was made in the skin approximately 1 to 2 mm lateral to the left sternal margin. The chest wall muscles were bluntly dissected layer by layer using curved forceps. The thorax was opened from the second and third intercostal spaces and then expanded with a chest expander, and the pericardium was resected. A 7-0 suture needle with thread was inserted through the anterior descending coronary artery and ligated 2 mm below the inferior margin of the left atrial appendage and 0.5 mm adjacent to the pulmonary artery cone. The suture was ligated with appropriate tension, controlling the needle insertion depth (preferably until the fine needle was barely visible) and the needle width (approximately 2 mm). Successful ischemia was confirmed when the outer surface of the anterior wall of the left ventricle turned pale. After the operation, the chest cavity was squeezed to remove the air and the skin was sutured with 5-0 sutures. The mouse was removed from the ventilator after it woke up.

[0117] 2. Mouse Cardiac Function Determination

[0118] Mouse cardiac function was assessed using VINNO small animal Doppler color Doppler ultrasound. Mice were anesthetized with isoflurane, their chests hair removed, and a coupling agent applied. The ultrasound probe was placed parasternal to the mouse, perpendicular to the sternum and pointing at the 10-11 o'clock position above the right shoulder. The probe was fine-tuned to locate the standard parasternal long-axis view, and the sampling line was moved to the center of the largest chamber of the left ventricle. An M-mode ultrasound image was recorded at this point. An eight-point measurement method was used to mark the anterior wall at end-diastole (outer), anterior wall at end-diastole (inner), posterior wall at end-diastole (inner), posterior wall at end-diastole (outer), anterior wall at end-systole (outer), anterior wall at end-systole (inner), posterior wall at end-systole (inner), and posterior wall at end-systole (outer). Left ventricular diastolic function parameters such as ejection fraction and fractional shortening were obtained.

[0119] 3. HE staining

[0120] The excised heart tissue was cleaned of blood with 0.9% saline and fixed in 4 mL of 4% paraformaldehyde for at least 24 hours. The tissue was removed from the fixative and placed in an embedding frame for dehydration using a gradient dehydrator, following the following steps: 75% ethanol for 4 hours, 85% ethanol for 2 hours, 90% ethanol for 2 hours, 95% ethanol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65°C for 1 hour, paraffin II melted at 65°C for 1 hour, and paraffin III melted at 65°C for 1 hour.

[0121] Remove the wax-soaked sample and place it in the hot plate sample slot. Remove the stainless steel embedding base, add paraffin, and place the tissue into the bottom of the embedding base according to the standard embedding direction. Move it to the pre-cooling table to fix it. Cover the embedding frame and place the embedded tissue on the cold table. After the paraffin is completely solidified, demold it. Load it into the paraffin slicer sample holder, adjust the fast forward key to make the wax block tangent to the blade, adjust the section angle, perform rough trimming to a complete observation section, and perform continuous sectioning with a thickness of 3μm. Place the continuous tissue sections in a 45℃ water bath to flatten them. Use an adhesive slide to pick up the sections, drain the water, place them in a slice rack, and bake them in a 60℃ incubator for at least 2 hours.

[0122] Immerse the baked sections in xylene I for 15 min, xylene II for 15 min, xylene III for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and distilled water for 5 min to complete dewaxing.

[0123] Immerse the sections in hematoxylin solution for 5 minutes, rinse off excess stain, immerse in differentiation solution for 5 seconds, rinse with water, immerse in bluing solution for 5 seconds, and rinse with water. Dehydrate the sections in 85% and then 95% ethanol, each for 5 minutes, and then immerse in eosin solution for 5 minutes.

[0124] The sections were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and anhydrous ethanol III for 5 min for dehydration, xylene I for 5 min, and xylene II for 5 min for transparency, and then sealed with neutral gum.

[0125] 4.Masson staining

[0126] The steps of fixing, dehydrating, embedding, slicing and dewaxing the heart tissue are the same as those for HE staining in Example 5. The slices are immersed in potassium dichromate solution overnight, for about 16 hours, and then washed with water. The slices are immersed in a dye solution of equal proportions of iron hematoxylin A solution and iron hematoxylin B solution, stained for 1 minute, washed with water, differentiated with 1% hydrochloric acid alcohol, and washed with water. The slices are immersed in Ponceau red solution for 5 minutes, and the excess dye is washed away with water. The slices are immersed in phosphomolybdic acid solution for 1 minute and directly immersed in aniline blue solution for 10 seconds. The slices are rinsed and differentiated in 3 cylinders of 1% glacial acetic acid for about 5 seconds each, and dehydrated in 3 cylinders of anhydrous ethanol for about 3 seconds each. The slices are placed in xylene I for 5 minutes, xylene II for 5 minutes to make them transparent, and sealed with neutral gum.

[0127] 5. Sirius Red Staining

[0128] The steps of fixation, dehydration, embedding, sectioning, and dewaxing of cardiac tissue were the same as those for HE staining in Example 5. The sections were immersed in Sirius red stain for 8 minutes, dehydrated in three cylinders of anhydrous ethanol for 10 seconds each, and then placed in xylene I for 5 minutes and xylene II for 5 minutes to clear the sections. The sections were then mounted with neutral gum.

[0129] 6. Detection of α-SMA, COL-1, FN1, and PTGS2 mRNA levels in cardiac tissue by real-time quantitative PCR

[0130] 20 mg of cardiac tissue from the infarcted area was weighed, and tissue RNA was extracted for real-time quantitative PCR. The subsequent steps of real-time quantitative PCR were the same as those in Example 2.

[0131] 7. Western Blot detection of α-SMA, COL-1, FN1 and PTGS2 protein levels in cardiac tissue

[0132] 20 mg of cardiac tissue from the infarcted area was weighed and RIPA lysis buffer (strong) containing protease inhibitors was added to extract tissue proteins. The subsequent Western Blot steps were consistent with those in Example 2.

[0133] This specific implementation is merely an explanation of the present invention and is not a limitation of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Use of the small molecule compound E229-0305 in the preparation of a drug for preventing or treating myocardial fibrosis, wherein the structural formula of the small molecule compound E229-0305 is: PubChem number: 328610468, Chinese name is 1-(2,3-dimethylphenyl)-4-(1-{pyrrole, 2-A]quinoxaline-4-yl}piperidine-3-carbonyl)piperazine, English name is 1-(2,3-DIMETHYLPHENYL)-4-(1-{PYRROLO[1,2-A]QUINOXALIN-4-YL}PIPERIDINE-3-CARBONYL)PIPERAZINE, molecular formula is C 26 H 33 N5O, relative molecular mass: 467.3g / mol.

2. The use according to claim 1, characterized in that The small molecule compound E229-0305 inhibits TGF-β1-induced cardiac fibroblast proliferation, migration and fibroblast-to-myofibroblast transformation; the small molecule compound E229-0305 inhibits the high expression of α-SMA, COL-1, FN1 and PTGS2 proteins and mRNA in TGF-β1-induced cardiac fibroblasts.

3. The use according to claim 1, characterized in that The small molecule compound E229-0305 improves the cardiac function of mice with myocardial infarction, increases the cardiac ejection fraction and short-axis shortening rate; the small molecule compound E229-0305 inhibits the expression of α-SMA, COL-1, FN1 and PTGS2 proteins and mRNA in the heart tissue of mice with myocardial infarction.

4. The use according to claim 1, characterized in that The drug uses the small molecule compound E229-0305 as an active ingredient, and its concentration is 1 nM to 1 mM.

5. The use according to claim 4, characterized in that The concentration of the small molecule compound E229-0305 is 5 to 20 μM.

6. The use according to claim 1, characterized in that The medicine may include excipients, which are one or more of diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavors.

7. The use according to claim 1, characterized in that The pharmaceutical dosage form is injection, tablet, powder injection, granule, capsule, oral solution, pill, gel, suppository, ointment, emulsion, mixture, suspension or cream.

8. The use according to claim 1, characterized in that The administration routes of the drug include oral administration, injection, respiratory inhalation, topical administration, sublingual administration and rectal administration.

9. The use according to claim 1, characterized in that The myocardial fibrosis includes myocardial fibrosis caused by myocardial infarction, diabetes, hypertension, myocarditis, valvular heart disease, heart failure, and coronary heart disease.

10. A pharmaceutical composition, characterized in that The invention comprises an effective dose of a small molecule compound E229-0305 as an active ingredient, wherein the structural formula of the small molecule compound E229-0305 is Its concentration is 1nM~1mM.

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