Application of chrysin-7-0-beta-D-glucuronide in treating ischemic heart disease or LRP6 inhibitor

Abstract

The invention discloses application of chrysin-7-0-beta-D-glucuronide in treatment of ischemic heart disease or an LRP6 inhibitor, and belongs to the technical field of treatment of ischemic heart disease. The invention provides a new application of a small molecular compound chrysin-7-0-beta-D-glucuronide, the compound realizes regulation and control on a cardiac muscle cell cycle by inhibiting LRP6, and finally promotes repair of cardiac muscle cells and inhibits apoptosis. According to the invention, by exploring the function of inhibiting LRP6 protein of a small molecule drug, a powerful repair reaction is guided after myocardial ischemia reperfusion, so that effective treatment of myocardial infarction is realized. Therefore, finding of an LRP6 inhibitor not only has a potential curative effect on promoting myocardial cells to enter a cell cycle, but also has important significance on treatment of MI.

Description

Technical Field

[0001] The present invention relates to the technical field of ischemic heart disease treatment, and in particular, to the use of chrysin-7-0-β-D-glucuronide in the treatment of ischemic heart disease or an LRP6 inhibitor. Background Art

[0002] Myocardial infarction (MI) is one of the leading causes of disability and death worldwide. Irreversible damage to early cardiomyocytes and secondary cell death can lead to permanent loss of cardiac contractility, which in turn develops into heart failure. However, adult mammalian cardiomyocytes have limited proliferation and regeneration capacity after injury.

[0003] In clinical practice, effective strategies for saving ischemic myocardium mainly focus on timely reperfusion to save dying myocardium, reduce the infarct area and improve cardiac function. Emergency treatment methods include thrombolytic therapy, coronary artery dredging and stent implantation, which can quickly restore the patient's blood supply, enable the patient to rebuild cardiac function to a large extent, and ensure the maintenance of normal life. However, these treatment methods have limitations, such as high invasiveness, thrombosis, and immune rejection. In addition, because early ischemia causes irreversible damage to myocardial cells, even after blood supply is restored, dying myocardial cells may continue to die due to previously accumulated necrotic factors, thereby expanding the scope of infarction.

[0004] In basic research, therapeutic strategies such as stem cell transplantation, exosomes, and myocardial patch have achieved varying degrees of success in treating myocardial infarction and promoting the recovery of cardiac function. Although these research methods have made some progress in regulating inflammation, promoting angiogenesis, and reducing ROS, the effective replenishment of lost myocardium has not yet reached the ideal level.

[0005] Clinical research on cardiac regeneration has mainly focused on cell-based therapies, including bone marrow-derived cells, mesenchymal stem cells, and putative cardiac progenitor cells. Although these studies have achieved replenishment of cardiomyocyte numbers, the lack of fusion between exogenous cardiomyocytes and native cardiomyocytes has resulted in minimal or even negligible effects of cardiomyocyte transplantation on cardiac function, a result that has prompted the search for new methods to promote cardiac regeneration and repair. Tani Hidenori et al. achieved cardiac reprogramming under conditions of chronic MI by using the Tcf21 / reporter / MGTH2A transgenic mouse system. This system can induce the expression of MGTH and reporter genes in cardiac fibroblasts (CFs), converting these CFs into induced cardiomyocyte-like cells (iCMs), thereby achieving cardiac regeneration. Under conditions of chronic MI, the research team found that approximately 2% of CFs were successfully converted into iCMs through in vivo cardiac reprogramming. These iCMs were not formed by a simple fusion mechanism, but were generated by true cardiac reprogramming, showing a new cardiomyocyte identity, which provides opportunities for the development of new therapies for chronic MI and heart failure. It is worth mentioning that many clinical trials of gene therapy have failed to achieve the expected results, which may be related to the mismatch of the concentration of the regulated gene and its product at the target site and the lack of understanding of the pathogenesis of cardiovascular disease. In addition, in the clinical practice of implementing gene therapy, safety factors should be considered, including potential off-target effects, tumorigenesis induced by certain viral vectors, and the occurrence of arrhythmias.

[0006] LRP6 (lipoprotein receptor-related proteins 6) has entered the researchers' field of vision as a key factor that regulates adult cardiomyocytes to re-enter the cell cycle and promote myocardial regeneration and repair. LRP6-related gene editing or drug inhibition may be a potential treatment for myocardial injury after MI. Since small molecule compounds have broad clinical translational application prospects, the development of specific inhibitors for LRP6 has important clinical application potential. In addition to myocardial injury, ventricular wall collapse and impaired conductivity are also common phenomena after MI.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide the use of chrysin-7-0-B-D-glucuronide in the treatment of ischemic heart disease or LRP6 inhibitors to solve the above technical problems.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides the use of chrysin-7-0-B-D-glucuronide and / or its salt in the preparation of LRP6 inhibitors.

[0011] In a second aspect, the present invention also provides the use of chrysin-7-0-B-D-glucuronide and / or its salts in the preparation of a medicament for treating ischemic heart disease and its complications.

[0012] In a third aspect, the present invention also provides the use of chrysin-7-0-B-D-glucuronide and / or its salt in the preparation of a reagent or drug for at least one of the following uses, wherein the reagent or drug is used for:

[0013] (1) Promote the proliferation of cardiomyocytes;

[0014] (2) Reduce cardiomyocyte apoptosis;

[0015] (3) inhibiting the expression of apoptosis-related proteins;

[0016] (4) Regulate the cell cycle;

[0017] (5) Promote the repair of ischemic myocardial tissue.

[0018] The present invention has the following beneficial effects:

[0019] (1) The present invention uses Discovery Studio software to analyze the three-dimensional structure of LRP6 protein downloaded from the PBD database, and uses LRP6 as a target to screen small molecule compounds with specific binding ability in the small molecule compound library of Taoshu Biology. After high-throughput small molecule drug screening of potential targets that promote myocardial cell proliferation and repair, and at the same time, verifying its therapeutic effect through biological means, it is confirmed that the small molecule compound chrysin-7-0-B-D-glucuronide achieves regulation of myocardial cell cycle by inhibiting LRP6, ultimately promoting myocardial cell repair and inhibiting cell apoptosis. These results provide strong support for the preparation of LRP6 inhibitors or the treatment of ischemic heart disease.

[0020] (2) Experiments on rats with myocardial ischemia-reperfusion have shown that chrysin-7-0-B-D-glucuronide plays a certain role in reducing myocardial infarction area and cardiac fibrosis, thereby improving rat cardiac function. This provides substantial support for the application of chrysin-7-0-B-D-glucuronide in the treatment of cardiovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 The expression of LRP6 protein in the infarct area of ​​myocardial ischemia-reperfusion model in adult SD rats; (a) qPCR was used to detect the expression of LRP6 in myocardial infarction-reperfusion heart tissue; (b) Western blot was used to detect the expression of LRP6 in myocardial infarction-reperfusion heart tissue; (c) Immunofluorescence (20×) was used to detect the expression of LRP6 in myocardial infarction-reperfusion heart tissue, scale = 100μm; images represent n = 3 independent repeated experiments; data in the figure represent Mean ± SD. Significant difference: **p < 0.01, ***p < 0.001;

[0023] Figure 2 The names and chemical structure diagrams of the top 10 small molecule compounds for high-throughput screening;

[0024] Figure 3 The result diagram of visualizing LRP6-chrysin-7-0-B-D-glucuronide with PyMOL software ((a) Molecular docking software shows the binding between LRP6 and chrysin-7-0-B-D-glucuronide; (b) Molecular docking software predicts the binding site and interaction force between LRP6 and chrysin-7-0-B-D-glucuronide);

[0025] Figure 4 The results of in vitro verification of the interaction between LRP6 and chrysin-7-0-B-D-glucuronide ((a) In vitro molecular interaction experiments were conducted with different concentrations of LRP6 and chrysin-7-0-B-D-glucuronide to detect the binding strength between the two; (b) the signal value two seconds before the termination of the injection was used to make an affinity fitting curve to analyze the affinity between the two);

[0026] Figure 5The experimental results of chrysin-7-0-B-D-glucuronide improving AC16 cell viability and promoting cell proliferation are shown in Figure 1 ((a) After treating AC16 cells with a specified concentration of chrysin-7-0-B-D-glucuronide for 24 hours, the proliferation of AC16 cells was determined by CCK8; (b) After treating cells with a specified concentration of chrysin-7-0-B-D-glucuronide (6 μM) for 24 hours, cell survival was detected by trypan blue staining. (b) After treating cells with a specified concentration of chrysin-7-0-B-D-glucuronide (6 μM) for 24 hours, cell survival was counted by YO-PRO-1 fluorescence staining, scale = 100 μm. The images represent n = 3 independent repeated experiments. The data in the figure represent Mean ± SD. Significant differences: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001);

[0027] Figure 6 The results of the experiment on the inhibition of apoptosis of AC16 cells by chrysin-7-0-B-D-glucuronide (a) after treating AC16 cells with a specified concentration of chrysin-7-0-B-D-glucuronide (6 μM) for 24 hours, the apoptosis of AC16 was detected by flow cytometry; (b) the statistical graph of the positive cell rate of flow cytometry results; (c) after treating AC16 cells with a specified concentration of chrysin-7-0-B-D-glucuronide (6 μM) for 24 hours, the apoptosis of AC16 was detected by apoptosis and necrosis staining, scale bar = 100 μm);

[0028] Figure 7 This is a graph showing changes in cell apoptosis-related indices detected by Western blot after cells were treated with chrysin-7-0-β-D-glucuronide;

[0029] Figure 8 The results of the experiment that chrysin-7-0-B-D-glucuronide promotes AC16 proliferation by regulating the cell cycle ((a) After treating AC16 cells with the specified concentration of chrysin-7-0-B-D-glucuronide (6 μM) for 24 hours, the cell cycle distribution of AC16 was detected by flow cytometry; (b) The statistical graph of the positive cell rate of the flow cytometry results; (c) Western blot detection of changes in cell cycle-related indicators after cells were treated with chrysin-7-0-B-D-glucuronide. The images represent n=3 independent repeated experiments. The data in the figure represent Mean±SD. Significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0030] Fig. 9The experimental results of chrysin-7-0-B-D-glucuronide acting on LRP6 protein to affect cycle-related proteins ((a) After treating AC16 cells with a specified concentration of chrysin-7-0-B-D-glucuronide (6 μM) for 24 hours, qPCR was used to detect the expression of cycle-related protein genes; (b) Western blot was used to detect the changes in cycle protein-related indicators after cells were treated with chrysin-7-0-B-D-glucuronide. The data in the figure represent Mean±SD. Significant differences: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0031] Fig.10 The results of the experiment on the therapeutic effect of chrysin-7-0-β-D-glucuronide on rats with myocardial ischemia-reperfusion ((a) M-mode echocardiographic images of different groups after 28 days of treatment; Doppler echocardiographic images of different groups after 28 days of treatment; (be) Quantitative comparison of left ventricular function of different groups after 28 days of treatment; (f) Masson trichrome staining of cardiac regional sections of different groups, scale = 1mm (myocardium is red, fibrous tissue is blue); (g) Quantitative analysis of the fibrosis area of ​​the anterior wall of the left ventricle based on Masson's trichrome staining images; (h) HE staining images of organs of rats in each group after 28 days of treatment. Scale = 500μm; Data in the figure represent Mean±SD; Significant difference: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

[0032] Fig.11LRP6 deficiency promotes cardiac functional recovery and induces sudden death associated with ventricular arrhythmia 4 weeks after myocardial infarction (MI). (A) Quantitative detection of serum cTnI in each group (n = 5). (B) Masson trichrome-stained cardiac slice images. (C) Quantitative analysis of infarct size in cardiac slices. (D) M-mode echocardiography (lower) and Doppler ultrasound (upper) were performed, and (E) E / A ratio, (F) left ventricular ejection fraction (LVEF), and (G) left ventricular fractional shortening (LVFS) were calculated (n = 5). (H) Representative optical mapping images of action potential and calcium transient duration in each group, and the activation time of (I) action potential and (J) calcium transient duration were recorded. Ventricular electrophysiological stability and VA susceptibility were evaluated by electrocardiography (ECG), and (K) VA inducibility and (L) VF threshold were measured. (M) Throughout the process, the mortality of mice was monitored and statistically analyzed, and the Kaplan-Meier survival curve of each group was obtained; n = 10 per group; the data in the figure represent mean ± standard deviation (Mean ± SD). Error bars represent standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, p values ​​were generated by one-way analysis of variance (ANOVA) and tested by Tukey's post hoc multiple comparison test;

[0033] Fig.12 The results of cardiac cycle detection (HR) in the LRP6 knockout mouse model after myocardial infarction, as well as the statistical results of the action potential duration at 90% repolarization (APD90) and the calcium transient duration at 90% recovery (CTD90) averaged in the optical mapping field after myocardial infarction; n = 5 mice per group in Figure A, ns means no significant difference; (B) in isolated rat hearts, the action potential duration at 90% repolarization (APD90) and (C) the calcium transient duration at 90% recovery (CTD90) averaged in the optical mapping field after myocardial infarction; significant differences are marked as: ****P<0.0001;

[0034] Fig.13 The amino acid sequence results of LRP6 proteins from different species. DETAILED DESCRIPTION

[0035] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0036]

[0037] In a first aspect, the present invention provides the use of chrysin-7-0-B-D-glucuronide and / or its salt in the preparation of LRP6 inhibitors.

[0038] The present invention uses Discovery Studio software to analyze the three-dimensional structure of LRP6 protein downloaded from the PBD database, and uses LRP6 as a target to screen small molecule compounds with specific binding ability in the small molecule compound library of Taoshu Biology. Through high-throughput small molecule drug screening of potential targets for promoting myocardial cell proliferation and repair, and at the same time, verifying its therapeutic effect through biological means, it is confirmed that the small molecule compound chrysin-7-0-B-D-glucuronide achieves the regulation of myocardial cell cycle by inhibiting LRP6, and finally promotes the repair of myocardial cells and inhibits cell apoptosis. These results provide strong support for the preparation of LRP6 inhibitors.

[0039] In the present invention, Chrysin-7-O-β-D-glucuronide (abbreviated as C7Og), i.e. Chrysin-7-O-glucuronide, has a molecular formula of C 21 H 18 O 10 , molecular weight is 430.36, CAS number is 35775-49-6.

[0040] The screening of LRP6 small molecule inhibitors is based on an in-depth understanding of the biological functions of LRP6, especially its key role in cardiomyocyte proliferation and myocardial repair. By analyzing the mechanism of action of LRP6, Chen's research team revealed the molecular pathways by which LRP6 regulates cardiomyocyte proliferation, especially through the regulation of the ING5 / P21 signaling pathway. Previously, LRP6 was mainly considered to be a co-receptor of the Wnt signaling pathway, but this invention revealed for the first time the new function of LRP6 in late cardiomyocyte proliferation. This discovery not only deepens the understanding of cardiac cell proliferation and regeneration, but also provides new targets and methods for the treatment of heart diseases, which has important clinical significance. In this mechanism, the present invention found that: (1) LRP6 protein is highly expressed in rat myocardial infarction reperfusion heart tissue, resulting in a lack of regulation of cardiomyocyte cell cycle activity. Inhibition of LRP6 protein can guide adult cardiomyocytes to re-enter the cell cycle and promote cardiomyocyte repair; (2) Specific small molecule inhibitors inhibit the function of LRP6 protein, regulate the cell cycle to resist cardiomyocyte apoptosis, thereby promoting the repair of ischemic myocardial tissue.

[0041] The present invention aims to guide a strong repair response after myocardial ischemia-reperfusion by exploring the function of small molecule drugs to inhibit LRP6 protein, thereby achieving effective treatment of myocardial infarction. Therefore, finding inhibitors of LRP6 not only has potential therapeutic effects in promoting myocardial cells to enter the cell cycle, but also has important significance for the treatment of MI.

[0042] In order to further verify the molecular docking of chrysin-7-0-B-D-glucuronide with LRP6 in detail, the present invention uses PyMOL software to display the relevant structure. The analysis results show that LRP6 wraps and tightly binds chrysin-7-0-B-D-glucuronide. In a preferred embodiment of the present invention, the binding sites of chrysin-7-0-B-D-glucuronide and LRP6 are alanine at position 666, leucine at position 753, leucine at position 838, glycine at position 795, threonine at position 797, tyrosine at position 800, methionine at position 877, aspartic acid at position 878 and arginine at position 886 of LRP6, forming multiple binding pockets.

[0043] In a preferred embodiment of the present invention, chrysin-7-0-B-D-glucuronide electrostatically adsorbs the 886th arginine on LRP6 (the mutual attraction between positive and negative charges forms a strong interaction), and chrysin-7-0-B-D-glucuronide forms hydrogen bonds with the 753rd leucine, the 838th leucine, and the 797th threonine on LRP6 through hydrogen atoms; chrysin-7-0-B-D-glucuronide forms covalent bonds with the 795th glycine and the 800th tyrosine on LRP6, and chrysin-7-0-B-D-glucuronide forms a π-Anion interaction with the 878th aspartic acid on LRP6; chrysin-7-0-B-D-glucuronide interacts through π-alkyl, so that the π electron cloud of its aromatic ring can interact with the non-polar carbon-hydrogen bonds on the carbon atoms in the alkyl groups of the 666th alanine and the 877th methionine. Through the above-mentioned binding sites and actions, chrysin-7-0-β-D-glucuronide and LRP6 form an efficient binding.

[0044] In a second aspect, the present invention also provides the use of chrysin-7-0-B-D-glucuronide in the preparation of a medicament for treating ischemic heart disease and its complications.

[0045] The inventors verified on rats with myocardial ischemia-reperfusion that chrysin-7-0-B-D-glucuronide can improve rat cardiac function by reducing myocardial infarction area and cardiac fibrosis, which provides substantial support for the application of chrysin-7-0-B-D-glucuronide in the treatment of cardiovascular diseases.

[0046] In a preferred embodiment of the present invention, the above-mentioned ischemic heart disease and its complications include but are not limited to any one of myocardial infarction, coronary heart disease, angina pectoris, arrhythmia, ventricular fibrosis, heart failure, myocardial ischemia, myocarditis, ischemic cardiomyopathy and atherosclerosis.

[0047] In a preferred embodiment of the present invention, the above application includes at least one of the following application modes:

[0048] (1) Promote the proliferation of cardiomyocytes;

[0049] (2) Reduce cardiomyocyte apoptosis;

[0050] (3) inhibiting the expression of apoptosis-related proteins;

[0051] (4) Regulate the cell cycle;

[0052] (5) Promote the repair of ischemic myocardial tissue;

[0053] (6) Reduce myocardial infarction area;

[0054] (7) Reduce cardiac fibrosis.

[0055] The aforementioned inhibition of the expression of apoptosis-related proteins includes but is not limited to: inhibition of the expression of Bcl-2, Bax, PARP and Caspase3 proteins.

[0056] In a preferred embodiment of the present invention, the above-mentioned regulation of the cell cycle includes: promoting the transition of the cell cycle G1-S phase of the cardiomyocytes; thereby promoting the proliferation of the cardiomyocytes.

[0057] In a preferred embodiment of the present invention, the above-mentioned regulation of the cell cycle includes regulation in the following manner: by affecting the expression level of cyclin.

[0058] In a preferred embodiment of the present invention, the above-mentioned application of influencing the expression level of cyclin includes at least one of the following application modes:

[0059] (1) Downregulate the expression of ALDH7A1 gene;

[0060] (2) upregulating the expression of TSPAN5 gene;

[0061] (3) down-regulating the expression of at least one protein selected from the group consisting of Cyclin D1, CDK4, pRB and E2F1 proteins;

[0062] (4) Upregulate the expression of CDK2 protein.

[0063] (5) downregulate the expression of ACOT7 protein;

[0064] (6) downregulate the expression of LAM5 protein;

[0065] (7) Upregulate the expression of ERBB2 gene.

[0066] In another embodiment, the above-mentioned medicine or pharmaceutical composition can be a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol, ointment, soft and hard gelatin capsule, suppository, sterile injection solution or sterile packaged powder injection. In the present invention, the active ingredient is prepared into a medicine or pharmaceutical composition so that it can release the active ingredient quickly, slowly or delayed after being administered to a subject, for example, the active ingredient can be mixed with a carrier, diluted with a carrier or encapsulated in a carrier. The carrier, excipient and diluent can be a carrier, excipient and diluent that acts as an active ingredient, or a carrier, excipient and diluent that acts as a medium.

[0067] In another embodiment, the drug or pharmaceutical composition in the above application can be a solid, semisolid or liquid material, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, resin, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water syrup, methylcellulose, methyl and propyl paraffin, talc, magnesium stearate or liquid paraffin.

[0068] In another embodiment, the drug or pharmaceutical composition in the above application may further include a lubricant, a wetting agent, an emulsifying and suspending agent, a preservative, a sweetener or a flavoring agent.

[0069] The drug in the above application is preferably administered by injection.

[0070] In a third aspect, the present invention also provides the use of chrysin-7-0-B-D-glucuronide and / or its salt in the preparation of a reagent or drug for at least one of the following uses, wherein the reagent or drug is used for:

[0071] (1) Promote the proliferation of cardiomyocytes;

[0072] (2) Reduce cardiomyocyte apoptosis;

[0073] (3) inhibiting the expression of apoptosis-related proteins;

[0074] (4) Regulate the cell cycle;

[0075] (5) Promote the repair of ischemic myocardial tissue.

[0076] The uses of the above reagents or drugs include but are not limited to: adding to the in vitro culture medium of cardiomyocytes, repairing ischemic myocardial tissue in vitro, inhibiting the expression of apoptosis-related proteins in vitro, and regulating the cell cycle.

[0077] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0078] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0079] Example 1

[0080] This example investigates the expression of LRP6 in the myocardial infarction area.

[0081] In the mouse heart, LRP6 protein can affect the proliferation and survival of cardiac progenitor cells, and ablation may lead to cardiac development defects and cause serious cardiac morphological defects. Studies have found that using gene knockout technology, the specific deletion of LRP6 in mouse cardiomyocytes can induce a strong regenerative response after MI, resulting in a reduction in myocardial infarction area and improved left ventricular contractile function. In view of this, this example aims to further explore the expression of LRP6 protein in the rat myocardial infarction reperfusion model. The rat hearts 24 hours after myocardial infarction reperfusion were tested to study the expression of LRP6 in the rat heart in detail. The results provide an important basis for understanding the role of LRP6 in the repair process of rat hearts.

[0082] 1. Experimental Materials

[0083] (1) Experimental animals

[0084] SPF Sprague Dawley (SD) rats (200-230 g), of either sex, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. To ensure the hygiene standards of the animals, these rats were kept under conditions with a 12-h light-dark cycle and provided with adequate water and food. All experiments involving animals were reviewed and approved by the Experimental Animal Ethics Committee of Sichuan Provincial People's Hospital.

[0085] (2) Experimental materials and reagents

[0086] Table 1 Main experimental materials and reagents

[0087]

[0088]

[0089] Table 1 Main experimental materials and reagents

[0090]

[0091] (3) Experimental instruments

[0092] Table 2 Main experimental instruments and equipment

[0093]

[0094] 2. Experimental Methods

[0095] A. Construction of rat myocardial ischemia-reperfusion model

[0096] (1) Anesthesia and endotracheal intubation surgery preparation: SD rats were anesthetized with isoflurane and endotracheal intubation was performed using a small animal ventilator to ensure respiratory stability.

[0097] (2) Chest exposure and thoracotomy: The pleura was exposed between the third and fourth ribs of the left chest of the rat.

[0098] (3) Coronary artery slip knot ligation: Use 6-0 suture to slip knot ligature the left anterior descending branch of the coronary artery 2 to 3 mm away from the left atrial appendage. Ensure that there is no obvious bleeding in the heart during the operation and keep the surgical area clean.

[0099] (4) Layer-by-layer suturing: The surgical area is sutured layer by layer using sutures to ensure that each tissue layer is tightly combined.

[0100] (5) Establishment of myocardial ischemia-reperfusion model: After 40 minutes of ischemia, the coronary artery ligature was removed.

[0101] B. Tissue Collection and Real-time Fluorescence Quantitative PCR

[0102] Table 3 Primers for different genes

[0103]

[0104] (1) Rat model establishment and treatment: 24 h after the model was established, the rats were killed using anesthetics and the hearts were collected for subsequent studies.

[0105] (2) Cardiac zoning and tissue collection: The heart was divided into infarcted area, marginal area, and normal area, and equal volume of tissue was collected from each area for subsequent experiments.

[0106] (3) TRIzol separation and RNA extraction: RNA was extracted from tissues in the infarct area, marginal area, and normal area using the TRIzol separation method. RNA was extracted using the SteadyPure Tissue Cell Small RNA Extraction Kit to obtain total RNA.

[0107] (4) cDNA synthesis: cDNA was synthesized using the QuantiTect reverse transcription kit.

[0108] (5) Real-time fluorescence quantitative PCR experiment: The real-time fluorescence quantitative PCR experiment was performed using the SYBR Green Pro Taq HS premixed qPCR kit. The reaction plate was placed in a fluorescence quantitative PCR instrument and a temperature gradient cycle was started, including denaturation, annealing, and extension steps. Finally, the fluorescence signal increase curve and the threshold cycle number (Ct value) of the PCR product were obtained.

[0109] C. Tissue Collection and Western Blotting

[0110] (1) Tissue collection and lysis: Collect different areas of cardiac tissue, cut into pieces, add pre-configured RIPA complete lysis buffer (RIPA: PMSF: phosphatase inhibitor = 100:1:1), and lyse on ice. Grind twice using a grinder, and determine the total protein content by BCA method after centrifugation. Use 5× loading buffer and an appropriate amount of RIPA lysis buffer to dilute the protein to 30 μg / μL, denature at 95°C for 10 min, prepare tissue protein, and store at -80°C for later use after aliquoting.

[0111] (2) SDS-PAGE electrophoresis: Prepare gels of corresponding specifications according to the instructions of the SDS-PAGE premixed rapid gel kit of Liji Biotechnology. Add protein samples and markers to the sample loading tank and adjust the electrophoresis voltage to complete the separation of protein bands.

[0112] (3) Transfer and blocking: Use the wet transfer method to transfer the membrane. After the transfer, block the membrane with milk powder. After 1 hour, wash the membrane with PBST for 5 minutes on a decolorizing shaker. Repeat 6 times.

[0113] (4) Immunoblotting: Place the blocked membrane in the pre-prepared primary antibody at 4°C overnight. After recovering the primary antibody, wash the membrane, add the pre-prepared secondary antibody solution, incubate at 37°C for 1 hour, and wash the membrane again with PBST.

[0114] (5) Development step: Prepare the developer according to the instructions of the ultrasensitive ECL chemiluminescence kit, place the PVDF membrane containing an appropriate amount of the developer in the gel imaging system, and develop the protein bands.

[0115] D. Tissue Collection and Immunofluorescence

[0116] (1) Heart collection, fixation and dehydration: The heart was collected according to the instructions in Section 2.3.2, and the obtained heart tissue was fixed in 4% paraformaldehyde for 24 hours, and dehydrated automatically using a tissue dehydrator.

[0117] (2) Paraffin embedding and sectioning: The heart was paraffin embedded and cut into sections with a thickness of 5 μm.

[0118] (3) Permeabilization and preliminary incubation: The heart slices were permeabilized with 0.5% Triton X-100 and then incubated with pure goat serum for 30 min at room temperature.

[0119] (4) Immunofluorescence experiment: Incubate with LRP6 primary antibody (concentration 1:500) at 4°C overnight, wash with PBS three times, 5 min each time. Then incubate with corresponding fluorescent secondary antibody at 37°C for 1 h. Wash again with PBS three times, 5 min each time.

[0120] (5) DAPI staining and sealing: Incubate with DAPI dye at 37°C for 20 min, add sealing solution to the slices, and cover with a coverslip for preservation.

[0121] (6) Observation and photography: Observe and photograph the heart sections under a fluorescence confocal microscope.

[0122] E. Cardiac troponin-t (cTnT) detection

[0123] According to the kit instructions, a quantitative ELISA kit (Abcam) was used to detect cTnT levels. After each model was successfully established, 0.5 mL of venous blood was collected to detect cTnT concentration.

[0124] F. Physiological index detection

[0125] Physiological parameters were measured by echocardiography. SD rats were mechanically ventilated under isoflurane anesthesia. Echocardiograms were recorded using the Visualsonics Ultra-High Resolution Small Animal Ultrasound Imaging System (Visualsonics, Toronto, Canada). Pulsed-wave Doppler echocardiography and M-mode echocardiography were used to detect mitral valve blood flow.

[0126] G. Histological Analysis of Heart Sections

[0127] Four weeks later, all animals were euthanized. The hearts were removed and cut into paraffin sections with a thickness of 5 μm. The histological characteristics of each organ were obtained according to the manufacturer's instructions. The histological characteristics of myocardial infarction were obtained by Masson's trichrome staining and HE staining according to the manufacturer's instructions. The thickness of the infarct area was measured using Image J software.

[0128] H. Electrical integration testing

[0129] Electrical integration testing was performed as described previously [1,2]. Briefly, an electrical stimulator and an electrocardiogram (ECG) recorder were used to assess the propagation of electrical signals in scar tissue. Scar tissue was harvested from rats after euthanasia, and 3 mm of healthy tissue was retained at each end of the scar. The two ends of the tissue were connected to the electrical stimulator and ECG recorder, respectively. The electrical stimulator parameters were set at 10 mV and 1 Hz. Four weeks later, signals were recorded for 5 min using a three-lead ECG and analyzed using Lab Chart software to determine the duration of the QRS complex. Arrhythmia induction was assessed according to a standard clinical electrical stimulation (PES) protocol. The epicardial activation between healthy and infarcted myocardium was mapped using a 64-channel electrophysiological mapping system (MappingLab). After the heart was isolated and Langendorff perfused, a 64-channel electrode array (8 × 8 grid, 0.45 mm pitch) was positioned at the border zone of healthy and infarcted tissue. Electrical stimulation (2 mV, 6 Hz) was applied to the epicardial surface beneath the left atrium. Action potential and Ca2+ activation time were determined by identifying the point of maximum negative slope. The inducibility of ventricular arrhythmias and ventricular fibrillation threshold were assessed using a bipolar electrode catheter sutured to the epicardium of the right ventricle. Programmed electrical stimulation was performed at an intensity twice the diastolic threshold using 8 consecutive S1-S1 stimuli with a cycle length of 330 ms, followed by additional stimulation (S2-S3-S4).

[0130] I. Statistical Analysis

[0131] All data were presented as mean ± standard deviation, and Fisher's test, nonparametric Mann-Whitney rank sum test and one-way analysis of variance were used for relevant experimental statistical analysis. In all statistical analyses, P<0.05 indicated that the difference was statistically significant, and significant differences were marked as *p<0.05, **p<0.01, ***p<0.001.

[0132] The experimental results showed that LRP6 expression was significantly upregulated in the infarcted myocardium after myocardial ischemia-reperfusion.

[0133] In order to detect the expression of LRP6 after myocardial ischemia-reperfusion, this example uses SD rats to construct a myocardial ischemia-reperfusion model. After 24 hours, the rat hearts were collected for qPCR, Western Blot and immunofluorescence detection, respectively, to analyze the expression of LRP6 in the myocardium after myocardial ischemia-reperfusion. First, the myocardial ischemia-reperfusion model was successfully constructed, and the myocardial tissues of the infarct (the ischemic region), the marginal (the ischemic marginal region) and the normal region (the non-ischemic region) were taken 24 hours after surgery to detect the mRNA level of LRP6 in the myocardial tissue of adult rats by qRT-PCR. It was found that compared with the normal region, the mRNA expression of LRP6 in the heart tissue of the infarct region (P=0.0007) was significantly upregulated ( Figure 1 This reflects the difference in expression of LRP6 at the gene transcription level. Secondly, Western Blot analysis further confirmed the results at the protein level. In the infarcted area, the expression level of LRP6 protein increased significantly ( Figure 1 b), compared with the marginal area and normal area, the difference is significant. Finally, through immunofluorescence detection, the localization and expression of LRP6 can be visually observed at the cellular level. The results showed that in the infarct area, the fluorescence signal of LRP6 protein was significantly stronger than that in other areas ( Figure 1 c), which is consistent with the above molecular level results. The above research results consistently show that under the condition of myocardial ischemia-reperfusion, the expression of LRP6 shows significant changes, suggesting that it is expected to become a potential target for the treatment of myocardial infarction repair.

[0134] According to the results of qPCR, Western blot and immunofluorescence, the expression of LRP6 protein in the infarct area was significantly increased in the myocardial ischemia-reperfusion model of adult SD rats. Since LRP6 protein is involved in myocardial cell proliferation, its reduced expression helps cells enter the cell cycle, thereby promoting myocardial cell proliferation. Therefore, highly expressed LRP6 protein may serve as a potential target for treating myocardial infarction and promoting repair. Finding inhibitors of LRP6 protein has important research significance for the treatment of myocardial ischemia-reperfusion.

[0135] To evaluate the effect of LRP6 on cardiac repair, we studied the LRP6 conditional knockout (LRP6 - / - ) mice (C57BL / 6JCya-Lrp6 em1flox / Cya). It was found that the level of cardiac troponin I (cTnI) in the blood of mice after MI injury was significantly lower than that of wild-type (WT) mice ( Fig.11 Middle A). LRP6 was expressed in 4 weeks after MI according to echocardiographic analysis.- / - The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS) and other cardiac function parameters of mice were significantly improved compared with WT mice ( Fig.11 D, E, F and G; Supplementary information, Fig.12 In addition, analysis of trichrome-stained heart sections revealed that LRP6 - / - The infarct size of mice was significantly reduced ( Figure 1 B and C in Figure 3), indicating that there is a significant correlation between the improvement of cardiac function and the reduction of infarct size. - / - Calcium transient duration and action potential were assessed in left ventricular cardiomyocytes from mouse hearts.

[0136] Calcium ions (Ca 2+ ) transient usually lasts for minutes to hours after MI, which may lead to prolonged activation of cardiomyocyte action potentials. Fig.11 As shown in H and I, after MI injury or sham operation, WT and LRP6 - / - The left ventricular (LV) action potential activation time in all groups showed a tendency to be prolonged, among which LRP6 - / - The extension of the action potential duration (APD(90)) was more significant than that of the sham group. Fig.12 Middle B). We then measured the calcium transient activation time after MI injury and the results showed that the WT group and LRP6 - / - The activation time of the two groups was longer than that of the sham operation group ( Fig.11 In addition, a longer calcium transient duration (CTD(90)) was observed in both groups after MI, with LRP6 - / - The increase in the group was significantly higher than that in the WT group ( Fig.11 Zhong H, J; Supplementary information, Fig.12 C). The prolonged action potential and calcium transient activation time led to a prolonged cardiomyocyte reentry period, which affected the contractile function of cardiomyocytes. The ventricular early activation (VA) sensitivity was further evaluated using programmed pacing, and the ventricular fibrillation (VF) threshold was measured by rapid pacing. MI significantly aggravated VA sensitivity, as manifested by a higher induction rate and a lower VF threshold, and LRP6 was significantly increased in both sham and MI surgery. - / - The changes in the group were more significant ( Fig.11 Importantly, increased VA-induced hyperactivity was associated with higher mortality ( Fig.11 These findings suggest that although LRP6 knockout may improve cardiac function to some extent, it still has significant limitations in resolving the disorder of action potential and calcium ion activity in cardiomyocytes after MI injury.

[0137] Example 2

[0138] In this example, LRP6 inhibitor screening was performed.

[0139] 1. Experimental Materials

[0140] (1) Experimental cell lines

[0141] The human cardiomyocyte cell line AC16 was purchased from the American Type Culture Collection (ATCC).

[0142] (2) Experimental software

[0143] Table 4 Experimental software

[0144]

[0145] (3) Experimental reagents

[0146] Table 5 Main experimental materials and reagents

[0147]

[0148] Table 5 Main experimental materials and reagents

[0149]

[0150]

[0151] (4) Experimental instruments

[0152] Table 6 Main experimental instruments and equipment

[0153]

[0154] (5) Preparation of commonly used reagents

[0155] Cell culture medium: 1% penicillin-streptomycin mixture, 10% fetal bovine serum (FBS), 89% culture medium (DMEM / F-12);

[0156] Escherichia coli culture medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL double distilled water, adjust the pH to 7.2 with 5 mol / L NaOH.

[0157] 2. The screening method for LRP6 inhibitors is as follows:

[0158] A. Discovery Studio software high-throughput screening

[0159] (1) Protein structure acquisition and preprocessing: The LRP6 protein structure was retrieved from the PDB database and downloaded in .mol2 format. A new project was created in the Discovery Studio 2019 Client software, the protein structure was imported, and visualization was performed. The protein was modified by deleting ligands and unnecessary structures, adding hydrogen atoms to the protein, performing energy minimization, and marking the active sites.

[0160] (2) Introduction and pretreatment of small molecule compound library: The small molecule compound library structure of Taoshu Biological is introduced, and hydrogen atoms are added to the small molecules, and salt removal, charge adjustment and other treatments are performed. The molecular force field is loaded to obtain the required 3D conformation of the small molecule compound by minimizing the energy.

[0161] (3) Molecular docking: In the Docking module, import the pre-processed LRP6 protein and small molecule compound library structure. Configure the Docking parameters and start the Docking module to achieve the docking of protein and small molecule ligand and the prediction of binding sites.

[0162] (4) Screening and analysis: Based on the screening criteria such as the best binding position and the lowest negative binding free energy, the most likely small molecule compounds are screened out with reference to the LibdockScore comprehensive evaluation parameters.

[0163] B. PyMOL software visualization of protein-small molecule compounds

[0164] (1) Loading protein-small molecule structure: Download the PDB file containing protein (LRP6) and small molecule from the PDB database, use drawing software (ChemDraw) to draw the three-dimensional structure of the small molecule compound, and ensure the balance and stability of the molecular structure. Use PyMOL software to open the PDB file and load the three-dimensional structure of the protein and small molecule.

[0165] (2) Select relevant chains and adjust the display mode: Select the protein chain related to the small molecule binding and use different display modes provided by PyMOL, such as wireframe, ball-and-stick, or surface mode, to clearly show the binding mode of protein and small molecule.

[0166] (3) Marking key residues and interactions: Mark key residues that interact with small molecules, such as amino acid residues.

[0167] (4) Adjust the display mode and save the results: Adjust the display mode of proteins and small molecules as needed to obtain a clear binding image. Perform interaction expressions or distance measurements in PyMOL and save the visualization results for subsequent analysis and presentation.

[0168] The results showed that through batch screening of Taoshu Biotechnology's small molecule compound library, a variety of small molecule compounds were successfully screened out, which can effectively bind to LRP6 protein in three-dimensional structure. According to the final docking score results, the most potential binding compounds were selected for subsequent biological verification. Table 7 lists the names, molecular formulas and docking scores of the top 10 compounds. Figure 2 Their chemical structures are shown.

[0169] Table 7 Top 10 small molecule compounds in high throughput screening

[0170]

[0171] The results of molecular docking analysis between Chrysin-7-O-glucuronide and LRP6 protein are as follows:

[0172] In order to further verify the molecular docking of Chrysin-7-O-glucuronide and LRP6 in detail, the relevant structure was displayed using PyMOL software. The analysis results showed that LRP6 wrapped and tightly bound Chrysin-7-O-glucuronide (see Figure 3 ).exist Figure 3 It was shown in the figure that Chrysin-7-O-glucuronide forms an efficient binding with LRP6, and its binding sites include alanine 666 (ALA), leucine 753, 838 (LEU), glycine 795 (GLY), threonine 797 (THR), tyrosine 800 (TYR), methionine 877 (MET), aspartic acid 878 (ASP), and arginine 886 (ARG), forming multiple binding pockets. In terms of interaction force, Chrysin-7-O-glucuronide and ARG form a strong interaction through the mutual attraction between positive and negative charges. Chrysin-7-O-glucuronide and ARG (886) form a strong binding pocket between ARG (886) and threonine 666 (ALA), leucine 753, 838 (LEU), glycine 795 (GLY), threonine 797 (THR), tyrosine 800 (TYR), methionine 877 (MET), aspartic acid 878 (ASP), and arginine 886 (ARG). In addition, Chrysin-7-O-glucuronide forms hydrogen bonds with the more negatively charged LEU and THR through hydrogen atoms, forms covalent chemical bonds with GLY and TYR, and forms π-Anion interactions with ASP. It is worth noting that Chrysin-7-O-glucuronide interacts with the non-polar carbon-hydrogen bonds on the carbon atoms in the ALA and MET alkyl groups through π-Alkyl interactions. The distances of these interactions are This docking analysis provides a detailed understanding of the binding mode of Chrysin-7-O-glucuronide to LRP6, providing an important reference for subsequent biological validation. Analysis of the amino acid sequences of human, porcine, rat and mouse LRP6 revealed that C7Og has highly consistent binding sites in these species ( Fig.13 ), Fig.13 The results showed that the amino acid sequence of the binding site of C7Og was the same as that of LRP6 in different species, and its binding site was highly conserved, indicating that it has a cross-species inhibitory effect.

[0173] Example 3

[0174] In this example, human LRP6 protein was recombinantly expressed and a surface plasmon resonance molecular interaction experiment was performed.

[0175] A. Recombinant protein expression and purification of LRP6 protein

[0176] (1) Gene cloning and expression: The sequence of the human LRP6 protein gene was retrieved and obtained using Pubmed, and cloned into the pET28a vector containing a 6×His tag. The plasmid was transformed into Escherichia coli BL21 and cultured in LB medium containing 50 μg / mL kanamycin until the OD value of the bacterial solution reached 0.6-0.8 at a wavelength of 600 nm. Protein expression was induced using 1 mM IPTG at 16°C for 6 h.

[0177] (2) Protein expression verification: Bacterial precipitates were collected, and the bacteria were lysed by ultrasonication in an ice bath. Then, loading buffer was added and boiled in a 95°C metal bath to denature the protein.

[0178] (3) Protein purification: Collect the bacterial precipitate in the culture, lyse, centrifuge and filter, and then adsorb LRP6 protein through a Ni-NTA purification column (Ni-NTA SefinoseTM Resin purification column). Wash with binding buffer 6 times to remove impurities. Elute the protein with 20 mM imidazole.

[0179] (4) Protein collection: Use an ultrafiltration tube to collect the eluate containing LRP6. Add loading buffer and boil in a 95°C metal bath to denature the protein. Use ELISA to cut out the band of the target protein on the stained Native-PAGE gel. Use a PAGE gel protein micro-recovery kit according to the instructions to collect LRP6 protein.

[0180] B. Surface plasmon resonance molecular interaction experiments

[0181] (1) Protein concentration determination: The protein concentration of the purified LRP6 protein was determined using a BCA kit to ensure accurate protein quantity.

[0182] (2) Biotinylation labeling: According to the steps in the G-MM-IGT Biotinylation Kit reagent manual, the LRP6 protein was biotinylated so as to be fixed to the chip.

[0183] (3) Chip coupling protein: After the SSA sensor is pre-wetted, the biotinylated LRP6 protein is placed on a 96-well plate. The solidification steps are edited in sequence according to the protein solidification operation process to couple the LRP6 protein to the biotinylated sensor to ensure a good coupling effect.

[0184] (4) Injection of small molecule compounds: small molecule compounds with different concentration gradients (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM) were prepared and injected. Reference wells were set and 200 μL of PBST + 1% DMSO buffer was added to each well.

[0185] (5) Data recording and fitting: The running program was set up according to the molecular interaction experiment steps. The baseline, association, and dissociation functions were used to detect the signal values ​​of small molecule binding to the protein at multiple concentrations. The Octet high-throughput analysis software was then used to analyze the results and perform data fitting to obtain the affinity constants of the small molecule compounds and the LRP6 protein.

[0186] In vitro experiments have identified the interaction between LRP6 and Chrysin-7-O-glucuronide. The results show that they exhibit fast binding and dissociation kinetics, and the binding strength is dose-dependent with the concentration of Chrysin-7-O-glucuronide (see Figure 4 ). Through calculation, the Kd value of LRP6 and Chrysin-7-O-glucuronide was determined to be 55.87nM, revealing that there is a very strong binding force between them (see Figure 4 Although the experimental results confirmed the high-affinity interaction between them, further studies are needed to gain a deeper understanding of their pharmacodynamic effects.

[0187] Example 4

[0188] This example experiments the effect of Chrysin-7-O-glucuronide on cardiomyocytes in vitro.

[0189] The experimental methods include:

[0190] A. Construction of myocardial ischemia-reperfusion model

[0191] (1) Cultivating cardiomyocytes: Select AC16 cell line and culture it in culture medium to ensure that AC16 cells maintain a healthy growth state.

[0192] (2) Ischemia-reperfusion treatment: The AC16 cell culture medium was replaced with serum-free culture medium, and then placed in a sealed box. The mixed gas culture method was used to exhaust the gas in the box by introducing N2 to ensure an anaerobic environment.

[0193] (3) Cell collection: When the O2 concentration is lower than 0.1%, the timing is started. After 1 h, the cells are removed and the cell culture medium is replaced with complete culture medium to simulate reperfusion conditions. Cell samples are collected for subsequent experiments.

[0194] B.CCK-8 and cell live / dead staining

[0195] (1) CCK-8 experiment: AC16 cells were seeded in a 96-well plate at 5×103 cells / well. After the cells reached an appropriate density (e.g., 8 hours), a myocardial ischemia-reperfusion model was constructed according to 3.3.5, and small molecule drugs were then added according to research needs. 24 hours after treatment, CCK-8 reagent was added to each well and incubated for 2 hours, and then the absorbance of each well was measured at 450 nm using an ELISA reader. Finally, statistical analysis was performed using Origin software to obtain quantitative results of cell viability.

[0196] (2) Trypan blue staining: AC16 cells were seeded in a 6-well plate at 2×105 cells / well. After the cell culture reached an appropriate density (e.g., 8 h), a cardiomyocyte ischemia-reperfusion model was constructed according to 3.3.5, and small molecule drugs were added according to research needs. 24 h after treatment, the cells were stained with a trypan blue kit. Then, the cell images were observed and photographed under an ordinary optical microscope. In order to quantitatively analyze the cell viability, ImageJ software was used to perform image analysis on the cells stained with trypan blue, and the number of live / dead cells was counted. Finally, statistical analysis was performed with the help of Origin software to obtain the quantitative results of cell viability.

[0197] (3) YO-PRO-1 staining: Similar to the trypan blue staining procedure, cells were stained using the YO-PRO-1 kit 24 hours after cell treatment. Subsequently, cell images were observed and photographed under a fluorescence microscope.

[0198] In order to verify the effect of Chrysin-7-O-glucuronide on cardiomyocytes, CCK-8 assay was performed to analyze cell proliferation in vitro. Figure 5Figure a shows that the effect of Chrysin-7-O-glucuronide on cell activity in ischemia-reperfusion is related to its concentration, and 6μM is determined to be the optimal dosing concentration for cells. Compared with the ischemia-reperfusion group, the cell viability of the 6μM Chrysin-7-O-glucuronide-treated group was enhanced by 26.47%±3.98%. Therefore, in subsequent experiments, 6μM Chrysin-7-O-glucuronide was selected as the optimal working concentration. Further trypan blue staining and YO-PRO-1 staining experiments confirmed the significant proliferation effect of 6μM Chrysin-7-O-glucuronide. Through the trypan blue staining experiment, it was clearly observed that in the Chrysin-7-O-glucuronide-treated group (concentration of 6μM) compared with the ischemia-reperfusion group, cardiomyocytes showed significant proliferation ( Figure 5 Middle b). YO-PRO-1 staining fluorescence was significantly reduced ( Figure 5 (c) indicates that the cells in the Chrysin-7-O-glucuronide-treated group underwent changes that may be related to the increase in proliferation activity. These results further support the role of Chrysin-7-O-glucuronide in promoting cardiomyocyte proliferation.

[0199] Example 5

[0200] In this example, Chrysin-7-O-glucuronide is used to regulate myocardial cell apoptosis. The method is as follows:

[0201] A. Flow cytometry to detect cell apoptosis

[0202] (1) Cell sample preparation: A myocardial cell ischemia-reperfusion model was constructed according to Example 4, and small molecule drugs were added. 100,000 cells were collected in a 1.5 mL centrifuge tube for each sample, and the supernatant was discarded after centrifugation. The cell pellet was resuspended in 990 μL of cell staining buffer.

[0203] (2) Staining: Add 5 μL Hoechst staining solution and 5 μL PI staining solution to each cell sample, resuspend thoroughly, and incubate on ice for 25 min.

[0204] (4) Flow cytometry and analysis: A flow cytometer was used to detect red fluorescence at an excitation wavelength of 638 nm and green fluorescence at 488 nm. FlowJo software was used to analyze the degree of cell apoptosis.

[0205] B. Detection of cell apoptosis by fluorescence microscopy

[0206] (1) Cell sample preparation: Prepare cells according to the method of step B of Example 4, and collect the suspended dead cells into a well plate using a desktop centrifuge.

[0207] (2) Staining: Add 900 μL of cell staining buffer, 5 μL of Hoechst staining solution, and 5 μL of PI staining solution to each well plate sample and incubate in an ice bath for 25 min.

[0208] (3) Microscope observation: After staining, the samples were washed once with PBS and then observed and photographed under a fluorescence microscope.

[0209] C. Protein Extraction and Western Blotting

[0210] (1) Prepare cells: Prepare cells for subsequent experiments according to the method of step B of Example 4.

[0211] (2) Protein extraction and Western blot: After completing cell preparation, first place the cells on ice and wash with PBS three times. Then, add RIPA complete lysis buffer (RIPA: PMSF: phosphatase inhibitor = 100:1:1), scrape the cells with a scraper, and transfer them to a centrifuge tube on ice. Let stand on ice for 30 minutes for ultrasonic disruption and lysis. Then, perform protein extraction according to the steps in 2.3.3 and perform Western blot experiments.

[0212] Apoptosis is an important mechanism of programmed cell death and is essential for maintaining the homeostasis of tissues and organs. The regulatory role of Chrysin-7-O-glucuronide in cardiomyocytes was deeply studied by flow cytometry apoptosis analysis. In the flow cytometry apoptosis analysis, the cells in the normal group showed an apoptosis rate of 8.21%±1.31%. The apoptosis rates of cells in the hypoxia-reperfusion group and the ischemia-hypoxia-reperfusion group increased to 10.59%±1.53% and 12.73%±1.28%, respectively. However, the ischemia-hypoxia-reperfusion group treated with Chrysin-7-O-glucuronide showed a significant apoptosis inhibition effect, and the apoptosis rate dropped to 7.94%±1.1%( Figure 6 Fluorescence microscopy results ( Figure 6 (c) The flow cytometry data were further verified to ensure the accuracy of the experimental results.

[0213] The subsequent Western Blotting experiment focused on the expression levels of apoptosis-related proteins. In the injury model, the total expression of caspase-3 and PARP did not change significantly, but after treatment with Chrysin-7-O-glucuronide, the expression of c-caspase-3 and c-PARP was significantly reduced. In addition, the expression of Bcl-2 and Bax proteins in the treatment group was significantly reduced compared with the injury group ( Figure 7 ). This series of molecular biological experiments proved that Chrysin-7-O-glucuronide played a significant anti-apoptotic effect by inhibiting the expression of apoptosis-related proteins.

[0214] Example 6

[0215] In this example, Chrysin-7-O-glucuronide was used to regulate the myocardial cell cycle. The method is as follows:

[0216] A. Flow cytometry to detect cell cycle

[0217] (1) Cell sample preparation: A myocardial cell ischemia-reperfusion model was constructed according to Example 4, and small molecule drugs were added. The cells were then digested with pancreatic enzymes, and the culture medium was washed and the cells were resuspended. After centrifugation, the cells were fixed in precooled 70% ethanol, and then washed and suspended to ensure that the cells were evenly dispersed.

[0218] (2) Preparation of propidium iodide staining solution: Prepare propidium iodide staining solution according to the instructions of the cell cycle detection kit.

[0219] (3) Staining: Add propidium iodide staining solution to each cell sample, resuspend thoroughly, and incubate at 37°C in the dark for 30 min.

[0220] (4) Flow cytometric detection and analysis: Green fluorescence and light scattering were detected using a flow cytometer at an excitation wavelength of 488 nm. Cellular DNA content and light scattering were analyzed using FlowJo software.

[0221] B. Protein extraction and protein immunoblotting (same as Example 5)

[0222] Studies have found that LRP6 protein can promote cardiomyocyte proliferation by regulating the cell cycle. In order to gain a deeper understanding of the effect of Chrysin-7-O-glucuronide on the AC16 cell cycle, flow cytometry experiments were performed. Figure 8The experimental results are shown in ab. In the normal group, the G1 phase accounted for 39.6% ± 1.01%, the G2 phase accounted for 18.93% ± 2.86%, and the S phase accounted for 36.27% ± 3.87%. In the hypoxia-reperfusion group, cells were mainly blocked in the G1 phase, while hypoxia-reperfusion caused more cells to be blocked in the G1 phase, accounting for 49.1% ± 2.79%. However, the hypoxia-reperfusion group treated with Chrysin-7-O-glucuronide showed a different cell cycle distribution, with the G1 phase accounting for 34.37% ± 1.33% and the S phase accounting for 40.43% ± 0.49%. This clearly shows that Chrysin-7-O-glucuronide treatment causes G1 phase cells to successfully enter the S phase. Therefore, it is concluded that Chrysin-7-O-glucuronide promotes the proliferation of AC16 cells by regulating the cell cycle by acting on the LRP6 protein.

[0223] In order to further confirm the regulatory effect of Chrysin-7-O-glucuronide on LRP6 protein, Western blot experiments were carried out. Through Western blot experiments, the expression level of the protein and possible post-translational modifications can be directly observed, further verifying the regulatory effect of Chrysin-7-O-glucuronide on LRP6 protein ( Figure 8 (c) Compared with the normal group, the expression of Cyclin D1 protein in the hypoxia-reperfusion and ischemia-hypoxia-reperfusion groups showed an upward trend. However, in the drug-treated ischemia-reperfusion group, the expression of Cyclin D1 protein decreased significantly, revealing the significant reversal effect of Chrysin-7-O-glucuronide treatment on Cyclin D1 protein expression. However, the expression level of CDK2 protein showed an opposite trend. Compared with the normal group, the expression of CDK2 protein in the hypoxia-reperfusion and ischemia-hypoxia-reperfusion groups showed a downward trend, while the expression of CDK2 in the drug-treated group increased. There was no significant difference in the expression of Cyclin A2 protein among the four groups. The WB experimental results were consistent with the flow cytometry results. This series of experimental results emphasizes that Chrysin-7-O-glucuronide has a significant effect on the regulation of the cardiomyocyte cell cycle by affecting the level of cyclin, thereby affecting the proliferation activity of cardiomyocytes.

[0224] Example 7

[0225] This example explores the specific mechanism by which Chrysin-7-O-glucuronide regulates cardiomyocyte proliferation through ALDH7A1 / TSPAN5 / ACOT7 proteins. The experimental method is real-time fluorescence quantitative PCR.

[0226] Table 8 Primer sequences of different genes

[0227]

[0228] (1) Cell sample collection: AC16 cells were cultured at 4×10 6 Cells / well were seeded in a 100 mm dish, and after the cells reached an appropriate density, a myocardial cell ischemia-reperfusion model was constructed according to Example 4, and small molecule drugs were added according to research requirements. Cell samples were collected 24 hours after treatment.

[0229] (2) Total RNA extraction: Use the total RNA extraction kit from Acryl and follow the instructions to extract total RNA. Use a micro-spectrophotometer to measure the RNA concentration and purity to ensure that the quality and concentration of the extracted RNA meet the experimental requirements.

[0230] (3) Reverse transcription: Use the Evo M-MLV reverse transcription premix kit to perform reverse transcription reaction and convert the extracted total RNA into corresponding cDNA.

[0231] (4) Real-time fluorescence quantitative PCR experiment: Set appropriate primer concentrations, set up the qRT-PCR reaction system and reaction conditions, use a fluorescence real-time quantitative PCR instrument to perform PCR reactions, and record the fluorescence signal. Obtain quantitative data on gene expression after small molecule drug treatment.

[0232] (5) Data analysis: GAPDH was used as the internal reference, and all results were standardized with GAPDH.

[0233] After Chrysin-7-O-glucuronide acted on LRP6 protein, the related genes were verified to reveal its regulatory mechanism on cardiomyocyte proliferation. The verification results showed that TSPAN5, ERBB2, LAM5, ACOT7 and ALDH7A1 genes changed after Chrysin-7-O-glucuronide acted on LRP6 protein. Fig. 9). Specifically, TSPAN5 and ERBB2 gene expression were upregulated, while LAM5, ACOT7, and ALDH7A1 gene expression were downregulated. Further analysis revealed that the regulatory function of ALDH7A1 in the cell cycle indicated that it plays a role in the transition from G1 to S phase, and Chrysin-7-O-glucuronide resulted in decreased ALDH7A1 expression. In addition, the study observed that TSPAN5 inhibited tumor growth by inhibiting the transition from the G1-S phase of the cell cycle in gastric cancer. The mechanism of action of Chrysin-7-O-glucuronide suggests that it promotes the transition from the G1-S phase of the cell cycle by upregulating TSPAN5, thereby promoting the growth of damaged myocardium. Upregulation of TSPAN5 led to increased expression of p27 and p15, while reducing the expression of Cyclin D1, CDK4, pRB, and E2F1, regulating cell growth and division. In addition, ACOT7 loss has been shown to induce cell cycle arrest by activating the p53-p21 signaling pathway without involving DNA damage response. ACOT7 is activated after myocardial cell injury, but after Chrysin-7-O-glucuronide, ACOT7 protein expression decreases, which may be a mechanism leading to myocardial cell proliferation. This series of findings indicates that Chrysin-7-O-glucuronide regulates cell cycle and related gene expression through the LRP6 protein-mediated signaling pathway, thereby affecting myocardial cell proliferation.

[0234] Example 8

[0235] This example explores the therapeutic effect of Chrysin-7-O-glucuronide on myocardial ischemia-reperfusion rats, constructs a rat myocardial ischemia-reperfusion model, and compares the treatment group with the sham group to evaluate the therapeutic effect of Chrysin-7-O-glucuronide. The performance of the composite material in the treatment process will be comprehensively examined through multiple evaluation methods such as rat echocardiography, cardiac function indicators, Masson staining, and HE staining.

[0236] 1. Experimental Materials

[0237] (1) Experimental animals

[0238] SPF Sprague Dawley (SD) rats (200-230 g), of either sex, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. To ensure the hygiene standards of the animals, these rats were kept under conditions with a 12-h light-dark cycle and provided with adequate water and food. All experiments involving animals were reviewed and approved by the Experimental Animal Ethics Committee of Sichuan Provincial People's Hospital.

[0239] (2) Experimental materials and reagents

[0240] Table 9 Main experimental materials and reagents

[0241]

[0242] (3) Experimental instruments

[0243] Table 10 Main experimental instruments and equipment

[0244]

[0245] 2. Experimental Methods

[0246] (1) Construction of myocardial ischemia-reperfusion model

[0247] Myocardial ischemia-reperfusion model was established in SD rats according to the experimental method of Example 1. After 40 minutes, the sham group was only subjected to thoracotomy without ligation of the left anterior descending artery. The PBS group and the drug-treated group were injected with PBS and Chrysin-7-O-glucuronide via tail vein injection, respectively.

[0248] (2) Detection of physiological indicators of myocardial infarction

[0249] Days later, the SD rats were anesthetized with isoflurane. After they entered deep anesthesia, they were detected using the Visualsonics ultra-high resolution small animal ultrasound imaging system. The mitral blood flow and cardiac function indicators were detected using pulsed Doppler ultrasound and M-mode ultrasound.

[0250] (3) Organ tissue collection, pathological HE staining and cardiac tissue Masson staining

[0251] (1) Tissue collection: After the 28-day treatment period, the rats were euthanized and the organ tissues were collected. In order to fix the tissues, 4% paraformaldehyde was used for fixation for more than 24 hours.

[0252] (2) Paraffin embedding and sectioning: Take an appropriate amount of tissue, dehydrate it, and then embed it in paraffin. Use a paraffin sectioning machine to cut the tissue into 5 μm slices, unfold the slices in warm water, and then place them on a slide.

[0253] (3) HE staining: The collected tissue samples are placed in a fully automatic staining machine for staining. After staining, the stained tissue sections are sealed with neutral gum to ensure the fixation and preservation of the samples. Finally, images are collected under a microscope.

[0254] (4) Masson staining: The heart tissue was placed in an automatic staining machine for dewaxing, and then stained according to the kit instructions provided by the Solebo manufacturer. After completion, the slides were sealed with neutral gum. Images were collected under an upright microscope.

[0255] (4) Statistical analysis

[0256] All data were presented as mean ± standard deviation, and Fisher's test, nonparametric Mann-Whitney rank sum test and one-way analysis of variance were used for relevant experimental statistical analysis. In all statistical analyses, P<0.05 indicated that the difference was statistically significant, and significant differences were marked as *p<0.05, **p<0.01, ***p<0.001.

[0257] 3. Experimental results and discussion

[0258] (1) Physiological indicators of rat heart

[0259] In order to evaluate the effect of multifunctional drug-loaded hydrogel on the recovery of cardiac function in rats with myocardial ischemia-reperfusion, a myocardial ischemia-reperfusion model was established in SD rats. Subsequently, PBS and Chrysin-7-O-glucuronide groups were respectively given by tail vein injection to explore the effect of composite drug-loaded hydrogel on the repair of myocardial injury in rats. Ultrasound examination was performed on the 28th day after treatment. M-mode echocardiography was used to detect cardiac systolic and diastolic function. Fig.10 Figure a shows that compared with the normal contraction of the left ventricular wall in the sham group, the contraction amplitude of the anterior ventricular wall in the PBS group was weak, and a certain contraction of the anterior ventricular wall was observed in the Chrysin-7-O-glucuronide group. Doppler ultrasound was used to detect cardiac function, and the results showed that the Chrysin-7-O-glucuronide injection group could improve the E / A peak ratio ( Fig.10 In addition, typical echocardiographic parameters such as ejection fraction (EF), fractional shortening (FS), and heart rate (HR) were analyzed ( Fig.10 b, c and e in Figure 2), it can be seen from the results that the treatment effect of the Chrysin-7-O-glucuronide injection group is significantly better than that of the PBS group. In summary, the results of echocardiography show that the Chrysin-7-O-glucuronide injection group can significantly improve the cardiac function of rats after myocardial ischemia-reperfusion.

[0260] (2) Masson staining of myocardial ischemia-reperfusion area confirmed the repair effect of Chrysin-7-O-glucuronide.

[0261] To further evaluate the role of Chrysin-7-O-glucuronide in myocardial injury repair, 28 days after treatment, three groups of different heart samples were collected, namely: sham group (sham operation group), PBS group (ischemia reperfusion group), and Chrysin-7-O-glucuronide group (drug group). Myocardial sections were processed by Masson staining to evaluate the repair effects of different groups. The staining results show (see Fig.10 f), due to the presence of a large amount of ischemic necrotic tissue in the myocardial ischemia-reperfusion area, a large amount of fibrous tissue appeared blue in the heart sections of the PBS group, and there was almost no red myocardial tissue in the anterior wall of the ventricle. However, some red myocardial tissue was visible in the damaged area of ​​Chrysin-7-O-glucuronide. The Masson staining images were processed using ImageJ software to calculate the thickness of the infarct wall, and there were significant differences among the groups (see Fig.10 (g in the figure). The thickness of the left ventricular wall in the PBS group was only 0.67±0.05 mm. These results indicate that Chrysin-7-O-glucuronide can enhance cardiac function and reduce scar formation in rats with myocardial ischemia-reperfusion, indicating that Chrysin-7-O-glucuronide has potential repair ability in myocardial injury repair.

[0262] (3) Effects of Chrysin-7-O-glucuronide treatment on organ tissue pathology.

[0263] In order to further explore the safety of Chrysin-7-O-glucuronide in vivo, after 28 days of treatment, the main organs (heart, liver, spleen, lung, and kidney) of rats in different treatment groups were stained with HE to observe their pathological changes. In the hematoxylin stain, the cell nucleus appears dark blue, while in the eosin solution, the cytoplasm appears red, which helps to observe the pathological changes of different organs through color changes. The experimental results are shown in Fig.10 In the h figure, the heart sections of the three groups showed neat arrangement of myocardial cells, and no characteristic changes of cardiac injury such as inflammation and fibrosis were observed in the interstitium. The hepatocytes were arranged divergently with the central vein as the center, and the nuclei were large and round. The spleen sections showed that the white bones were stained blue-purple, the dense red tissue formed trabeculae by the spleen capsule, and the rest was red pulp. The overall structure was intact and there was no abnormality. The lung tissue structure was intact, the glomerular structure was good, the blood vessels were not ruptured, and there was no obvious abnormality. The kidney sections showed that the glomerular structure was clear and complete, and no fibrosis was observed. There were no obvious abnormalities in the main organs of SD rats in each group, indicating that Chrysin-7-O-glucuronide is a relatively safe drug with excellent in vivo safety and biocompatibility.

[0264] In summary, the present invention has the following beneficial effects:

[0265] (1) Chrysin-7-O-glucuronide is a small molecule inhibitor of LRP6: Through the screening of LRP6 inhibitors, Chrysin-7-O-glucuronide was successfully confirmed as an effective LRP6 protein regulator. Molecular docking analysis revealed the binding site and binding mode of Chrysin-7-O-glucuronide to LRP6. The results of in vitro surface plasmon resonance molecular interaction experiments verified the direct interaction between Chrysin-7-O-glucuronide and LRP6, providing a theoretical basis for subsequent experiments.

[0266] (2) Chrysin-7-O-glucuronide promotes cardiomyocyte proliferation and inhibits apoptosis: The results of CCK-8, trypan blue staining and YO-PRO-1 staining further proved that Chrysin-7-O-glucuronide significantly promoted the proliferation of cardiomyocytes. Flow cytometry and related Western blot experiments revealed the inhibitory effect of Chrysin-7-O-glucuronide on cardiomyocyte apoptosis, providing important experimental support for its application in the treatment of heart diseases.

[0267] (3) Chrysin-7-O-glucuronide promotes cardiomyocyte proliferation by regulating the cardiomyocyte cell cycle: Flow cytometry, Western blot and qPCR experiments deeply analyzed the regulatory effect of Chrysin-7-O-glucuronide on the cardiomyocyte cell cycle, providing strong evidence for a more comprehensive understanding of its protective mechanism in cardiomyocytes. This series of results demonstrates the prospect of the small molecule compound Chrysin-7-O-glucuronide as a potential therapeutic drug for ischemic heart disease.

[0268] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0269] References:

[0270] 1.Liu,W.,et al.,Injectable Hydrogels Encapsulating Dual-FunctionalAu@Pt Core-Shell Nanoparticles Regulate Infarcted Microenvironments andEnhance the Therapeutic Efficacy of Stem Cells through Antioxidant andElectrical Integration.ACS Nano,2023.17(3):p.2053-2066.

[0271] 2.Zhou,L.,et al.,Wireless Self-Powered Optogenetic System for Long-Term Cardiac Neuromodulation to Improve Post-MICardiac Remodeling andMalignant Arrhythmia.Adv Sci(Weinh),2023.10(9):p.e2205551。

Claims

1. Use of chrysin-7-0-β-D-glucuronide and / or its salt in the preparation of LRP6 inhibitors.

2. The use according to claim 1, characterized in that: The binding sites of chrysin-7-0-B-D-glucuronide and LRP6 are alanine at position 666, leucine at position 753, leucine at position 838, glycine at position 795, threonine at position 797, tyrosine at position 800, methionine at position 877, aspartic acid at position 878 and arginine at position 886 of LRP6.

3. The use according to claim 2, characterized in that: The chrysin-7-0-B-D-glucuronide is electrostatically adsorbed with the 886th arginine on LRP6, and the chrysin-7-0-B-D-glucuronide forms hydrogen bonds with the 753rd leucine, the 838th leucine, and the 797th threonine on LRP6 through hydrogen atoms; the chrysin-7-0-B-D-glucuronide forms covalent bonds with the 795th glycine and the 800th tyrosine on LRP6, and the chrysin-7-0-B-D-glucuronide forms a π-Anion interaction with the 878th aspartic acid on LRP6; the chrysin-7-0-B-D-glucuronide interacts through a π-alkyl interaction, so that the π electron cloud of the aromatic ring can interact with the non-polar carbon-hydrogen bonds on the carbon atoms in the alkyl groups of the 666th alanine and the 877th methionine.

4. Use of chrysin-7-O-B-D-glucuronide and / or its salts in the preparation of medicines for treating ischemic heart disease and its complications.

5. The use according to claim 4, characterized in that: The ischemic heart disease and its complications are selected from any one of myocardial infarction, coronary heart disease, angina pectoris, arrhythmia, ventricular fibrosis, heart failure, myocardial ischemia, myocarditis and atherosclerosis.

6. The use according to claim 4, characterized in that: The application includes at least one of the following application modes: (1) Promote the proliferation of cardiomyocytes; (2) Reduce cardiomyocyte apoptosis; (3) inhibiting the expression of apoptosis-related proteins; (4) Regulate the cell cycle; (5) Promote the repair of ischemic myocardial tissue; (6) Reduce myocardial infarction area; (7) Reduce cardiac fibrosis.

7. The use according to claim 6, characterized in that: The cell cycle regulation includes: promoting the transition of the cell cycle G1-S phase of cardiomyocytes.

8. The use according to claim 6, characterized in that: The cell cycle regulation includes the following methods: regulating by affecting the expression level of cyclin.

9. The use according to claim 8, characterized in that: The method of influencing the expression level of cyclin includes at least one of the following application methods: (1) Downregulate the expression of ALDH7A1 gene; (2) upregulating the expression of TSPAN5 gene; (3) down-regulating the expression of at least one protein selected from the group consisting of Cyclin D1, CDK4, pRB and E2F1 proteins; (4) Upregulation of CDK2 protein expression; (5) downregulate the expression of ACOT7 protein; (6) downregulate the expression of LAM5 protein; (7) Upregulate the expression of ERBB2 gene.

10. Use of chrysin-7-O-B-D-glucuronide and / or its salt in the preparation of at least one of the following reagents or medicines, characterized in that: The reagent or drug is used for: (1) Promote the proliferation of cardiomyocytes; (2) Reduce cardiomyocyte apoptosis; (3) inhibiting the expression of apoptosis-related proteins; (4) Regulate the cell cycle; (5) Promote the repair of ischemic myocardial tissue.

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