Application of fructose-1, 6-diphosphate in preparation of medicine for preventing or treating congenital heart disease

Abstract

The invention relates to application of fructose-1, 6-diphosphate in preparation of a medicine for preventing or treating congenital heart disease. Experiments find that F-1 and 6-BP as a metabolism regulator can effectively relieve myocardial cell proliferation ability decline and glycolysis dysfunction caused by PFKP gene deletion so as to improve myocardial dysplasia, and when F-1 and 6-BP are used for preventing and treating congenital heart diseases, adverse reactions on maternal bodies and fetuses are few, and the F-1 and 6-BP have no toxic or side effects on the maternal bodies and the fetuses. Meanwhile, high risks caused by surgical treatment can be avoided through effective drug treatment.

Description

Technical Field

[0001] The present disclosure relates to the technical field of biomedicine, and in particular to an application of fructose-1,6-diphosphate in the preparation of a drug for preventing or treating congenital heart disease. Background Art

[0002] Embryonic myocardial dysplasia is one of the important pathological bases of congenital heart disease. Embryonic myocardial dysplasia not only leads to congenital heart disease, but may also directly cause intrauterine failure of congenital heart disease. At present, the intervention of congenital heart disease mainly relies on some known drug interventions, such as improving the metabolic state of cardiomyocytes by regulating metabolic pathways, but these existing drugs are not specific enough, which may cause side effects and have limited therapeutic effects on embryonic myocardial dysplasia. Secondly, the use of gene editing technology (such as CRISPR / Cas9, etc.) to genetically modify embryonic cells to correct gene mutations that may cause congenital heart disease in embryonic treatment still faces ethical controversy, and the technical operation is complex, and potential off-target effects may bring new health risks. In addition, stem cell therapy, which repairs damaged myocardial tissue through stem cell transplantation, affects the treatment effect and safety due to the limited source of stem cells and the existence of immune rejection reactions. Prenatal screening of the fetus through techniques such as echocardiography to detect early signs of congenital heart disease is mainly used for diagnosis, but lacks effective treatment measures and cannot directly improve embryonic myocardial maldevelopment. Although supplementing specific nutrients during pregnancy, such as folic acid, has a certain preventive effect, it lacks specificity and cannot directly act on the key metabolic pathways of myocardial development.

[0003] The prior art CN117899124A involves the use of Lactobacillus plantarum 24-7 in the postoperative maintenance treatment of congenital heart disease, but the patent is mainly aimed at postoperative treatment, which is different from the focus of embryonic intervention. At present, no relevant patent applications or research reports on the use of F-1,6-BP to prevent or treat embryonic myocardial dysplasia and in utero heart failure caused by congenital heart disease have been found. Summary of the invention

[0004] In order to solve the above technical problems, the present disclosure provides the use of fructose-1,6-bisphosphate (F-1,6-BP) in the preparation of a drug for preventing or treating congenital heart disease.

[0005] Studies have shown that abnormal glycolysis function is the main cause of decreased myocardial cell proliferation and myocardial dysplasia. F-1,6-BP has the characteristics of cell protection, metabolic support, anti-oxidation, anti-inflammatory and immunomodulatory. F-1,6-BP is an important metabolite in the middle and downstream of the glycolysis pathway. Fructose-6-phosphate kinase 1 (PFK1) catalyzes the conversion of fructose-6-phosphate (F6P) to F-1,6-BP. F-1,6-BP undergoes a series of reactions to generate phosphoenolpyruvate (PEP) and pyruvate, which participate in glucose metabolism. The loss of the PFKP gene will lead to a decrease in the level of F-1,6-BP in the glycolysis function, thereby leading to a decrease in the proliferation ability of myocardial cells, affecting the development of the embryonic myocardium, and then causing the occurrence of congenital heart disease and even the occurrence of intrauterine heart failure of congenital heart disease. The present invention discloses, through the construction of mouse models, selection experiments for optimizing different treatment doses, etc., that supplementation of F-1,6-BP can increase the level of glycolysis, improve the proliferation capacity of cardiomyocytes, and rescue embryonic myocardial dysplasia and congenital heart disease in utero heart failure caused by PFKP deficiency.

[0006] F-1,6-BP can be used by cells as an energy substrate, thereby improving cell energy metabolism. During embryonic myocardial development, this metabolic support helps the normal development and function maintenance of myocardial cells, especially when facing metabolic stress, such as hypoxia or malnutrition. As a natural metabolite, F-1,6-BP has good biocompatibility and safety in the body. The use of F-1,6-BP to prevent and treat congenital heart disease has fewer adverse reactions to the mother and fetus. At the same time, effective drug treatment can also avoid the high risks of surgical treatment.

[0007] In some embodiments of the present disclosure, fructose-1,6-diphosphate is used in the preparation of a drug for preventing or treating congenital heart disease.

[0008] In some embodiments of the present disclosure, fructose-1,6-bisphosphate is used in the preparation of a drug for preventing or treating embryonic myocardial dysplasia.

[0009] In some embodiments of the present disclosure, fructose-1,6-diphosphate is used in the preparation of a drug for preventing or treating intrauterine heart failure due to congenital heart disease.

[0010] In some embodiments of the present disclosure, fructose-1,6-bisphosphate is used in the preparation of a drug for alleviating glycolytic dysfunction.

[0011] In some embodiments of the present disclosure, fructose-1,6-bisphosphate is used in the preparation of a drug for improving the proliferation ability of cardiomyocytes.

[0012] In some embodiments of the present disclosure, the drug includes fructose-1,6-diphosphate and a pharmaceutically acceptable excipient or carrier.

[0013] In the present disclosure, the drug provides sufficient energy and biosynthetic precursors for cardiomyocytes by restoring the glycolytic function, thereby promoting the proliferation of cardiomyocytes and improving myocardial dysplasia.

[0014] Compared with the prior art, the technical solution provided by the embodiments of the present disclosure has the following advantages:

[0015] The fructose-1,6-bisphosphate provided by the present invention can restore glycolytic function and promote cardiomyocyte proliferation, effectively improve myocardial dysplasia caused by PFKP gene defects, and provide a new potential target for the treatment of congenital heart disease; it can play a dual role in the prevention and treatment of congenital heart disease when applied in drugs, and through early intervention, it can improve the development environment of embryonic cardiomyocytes and reduce the risk of congenital heart disease; it has high safety and few side effects in clinical application, and is suitable for use in the embryonic and fetal periods, avoiding the high risks brought by surgical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0018] Figure 1 Add glycolysis product F-1-6-BP to the hESC-CMs cell model to replenish the experimental time nodes and flow chart;

[0019] Figure 2 The results of immunofluorescence staining of proliferation marker Ki67 with the addition of glycolysis product F-1-6-BP to different groups of cell models;

[0020] Figure 3 This is a statistical diagram of quantitative analysis of Ki67 immunofluorescence staining in different groups of cell models;

[0021] Figure 4 Flow cytometric analysis of Ki67, a proliferation marker, with the addition of glycolysis product F-1-6-BP to different groups of cell models;

[0022] Figure 5These are the heart HE-stained sections of different groups of mouse models at embryonic day 17.5;

[0023] Figure 6 These are images of Ki67, a cardiac immunofluorescence proliferation marker, in different groups of mouse models at embryonic day 17.5;

[0024] Figure 7 The results of quantitative analysis of the thickness of the compact layer of the left and right ventricular walls of different groups of mouse models;

[0025] Figure 8 This is a statistical chart of quantitative analysis of Ki67 immunofluorescence staining in different groups of mouse models. DETAILED DESCRIPTION

[0026] In order to more clearly understand the above-mentioned objectives, features and advantages of the present disclosure, the scheme of the present disclosure will be further described below. It should be noted that the embodiments of the present disclosure and the features in the embodiments can be combined with each other without conflict.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present disclosure, but the present disclosure may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0028] Example 1

[0029] Building the Model

[0030] (1) Construction of a PFKP conditional knockout (cKO) mouse model: A conditional knockout (cKO) mouse model was generated using Myh6-driven Cre to specifically knock out PFKP in cardiomyocytes.

[0031] (2) Construction of human cardiomyocyte (hESC-CMs) model: PFKP gene knockout human embryonic stem cell model (hESC).

[0032] Example 2

[0033] Effects of F-1,6-BP on hESC-CMs (human embryonic stem cell-derived cardiomyocytes) in vitro

[0034] In vitro, 5 mM F-1,6-BP was added to WT and PFKP KO cardiomyocytes on day 13 of differentiation, and the proliferation of cardiomyocytes was evaluated by Ki67 immunofluorescence staining and flow cytometry analysis on day 15 of differentiation.

[0035] Experimental grouping: (1) WT+PBS: As a control group, it is used to evaluate the normal state of wild-type cells or tissues under normal physiological conditions (without any intervention). Provide a baseline data for comparing changes in other treatment groups. (2) WT+F-1,6-BP: Evaluate the effect of exogenous fructose-1,6-bisphosphate (F-1,6-BP) on wild-type cells or tissues. Determine whether F-1,6-BP will have some effect on cells or tissues (such as metabolic changes, cell proliferation, etc.) under a normal physiological background. If there is a significant difference between the WT+F-1,6-BP group and the WT+PBS group, it means that F-1,6-BP itself has some biological activity. (3) PFKP KO+PBS: Evaluate the effect of PFKP gene knockout on cells or tissues, without involving other interventions. Provide the basic phenotype after PFKP gene knockout for subsequent comparison. By comparing with the WT+PBS group, it can be clarified whether PFKP gene knockout has led to a specific phenotypic change (such as decreased cell proliferation ability, metabolic abnormalities, etc.). (4) PFKP KO+F-1,6-BP: Evaluate whether exogenous fructose-1,6-bisphosphate can affect the phenotype after PFKP gene knockout. By comparing with the PFKP KO+PBS group, it can be determined whether F-1,6-BP can improve or reverse the abnormal phenotype caused by PFKP gene knockout (such as restoring cell proliferation ability, improving metabolic defects, etc.). If there is a significant difference between the PFKP KO+F-1,6-BP group and the PFKP KO+PBS group, it means that F-1,6-BP may have an intervention effect on the phenotype of PFKP gene knockout.

[0036] 1. Immunofluorescence Staining

[0037] Immunofluorescence staining was used to evaluate the pluripotency of hESCs and the biological indicators of the structure and function of cardiomyocytes. (1) Cell culture slides: Place the slides in a 12-well or 24-well cell culture plate, pre-coated with matrix gel and placed in a constant temperature cell culture incubator (5% CO). 2, 37°C) to overnight. Determine the density of cell inoculation on the glass slide according to different experimental purposes. Inoculate the cells to be immunofluorescently stained on the glass slide at the required density, and stop culturing when the cells grow to the required density; (2) Aspirate the cell culture supernatant and wash the glass slide 3 times with a PBS solution without calcium and magnesium, each time for 5 minutes; (3) Fixation: Aspirate the PBS solution and fix with a 4% paraformaldehyde solution at room temperature for 20-30 minutes; (4) Aspirate the paraformaldehyde solution and wash the glass slide 3 times with a PBS solution without calcium and magnesium, each time for 5 minutes; (5) Permeabilization: Aspirate the PBS solution and permeabilize with a 0.1-0.3% concentration of Trion X-100 solution at room temperature for 10-15 minutes (if the protein to be tested is a cell membrane protein, this step can be skipped); (6) Fix the Trion Aspirate the X-100 solution and wash the slides 3 times with a PBS solution that does not contain calcium and magnesium, each time for 5 minutes; (7) Blocking: Aspirate the PBS solution and block with a 3-5% concentration of BSA solution at room temperature for 30 minutes; (8) Prepare the primary antibody: Calculate the required amount of antibody according to the number of slides. Refer to the instructions for use of each antibody, dissolve a certain amount of primary antibody in a 3-5% concentration of BSA solution, mix well and place in a 4°C refrigerator for standby use; (9) Primary antibody incubation: Add the above-prepared primary antibody dilution dropwise to the slide wrapped with sealing film. The amount of dropwise addition should be appropriate to infiltrate the cells on the slide, generally 50-100 μL. Use tweezers to carefully transfer the blocked slide to the primary antibody dropwise addition site on the slide, so that the cell side of the slide faces the primary antibody dilution to avoid the generation of bubbles that affect antibody binding. Place the slide in a light-proof humidified box and incubate overnight in a 4°C refrigerator. (10) Carefully pick up the glass slide with tweezers and transfer it to the cell culture plate, making sure that the cell side of the glass slide faces upward. Wash the glass slide three times with a PBS solution that does not contain calcium or magnesium, each time for 5 minutes; (11) Prepare the secondary antibody: Refer to the instructions for use of each antibody, dissolve a certain amount of the secondary antibody corresponding to the primary antibody in a 3-5% concentration of BSA solution, mix well, and avoid light. Place in a 4°C refrigerator for later use; (12) Incubate with the secondary antibody: Add the prepared secondary antibody dilution solution dropwise to the glass slide covered with sealing film. The amount added should be sufficient to wet the cells on the glass slide, generally 50-100 μL. Use tweezers to carefully transfer the glass slide to the secondary antibody addition site on the slide, with the cell side of the glass slide facing the secondary antibody dilution solution to avoid bubbles that may affect antibody binding. Place the glass slide in a light-proof humidified box and incubate at 37°C in the dark for 1 hour; (13) Use tweezers to carefully pick up the glass slide and transfer it to the cell culture plate, making sure the cell side of the glass slide faces upward. Wash the glass slide three times with a PBS solution that does not contain calcium or magnesium, each time for 5 minutes; (14) Seal the slide: Add anti-fluorescence attenuation sealing agent containing DAPI on the slide. The amount added should be enough to wet the cells on the slide.Use tweezers to carefully transfer the glass slide to the sealing medium drop site on the slide, with the cell surface of the glass slide facing the sealing medium to avoid the formation of bubbles. Place the slide in a light-proof humidified box and incubate in the dark at room temperature for 20-30 minutes or store in a refrigerator at 4°C for one week; (15) Set various parameters of the confocal microscope, observe, and take photos for preservation.

[0038] 2. Flow cytometry (FCM)

[0039] Use flow cytometry to evaluate the purity, differentiation efficiency and cell size of hESC cardiomyocytes. (1) Take out the cells for the next experiment from the cell culture incubator and observe the cell status under a microscope. Digest the cells to be tested by flow cytometry according to the method of cardiomyocyte inoculation (the amount of cells in each flow tube should not be less than 1×104); (2) Centrifuge at 1000r / min at room temperature for 5 minutes, discard the supernatant, resuspend and fix the cell pellet in the tube with 4% paraformaldehyde solution, and place at room temperature for 15-20 minutes (3) Centrifuge at room temperature for 5 minutes again, discard the supernatant, add PBS solution to wash the cells, and transfer the cells to a 1.5ml EP tube for the next step; (4) Centrifuge at room temperature for 5 minutes, discard the supernatant, and use 500uL of 1% Triton X-100 was used to resuspend and punch the cell pellet and placed at room temperature for 10 minutes; (5) centrifuged at room temperature for 5 minutes, the supernatant was discarded, PBS solution was added to wash the cells, and the cell pellet was resuspended and blocked with 3% BSA solution and placed at room temperature for 30 minutes; during the blocking process, 3% BSA solution was used to prepare the primary immunofluorescence antibody at a concentration of 1:100; 500uL of cells were prepared in each tube; (6) the cell suspension in step 4 was equally divided into two parts, centrifuged at room temperature for 5 minutes, the supernatant was aspirated, one of the parts was resuspended with the pre-prepared cTnT mouse monoclonal antibody (or other types of cardiomyocyte-specific antibodies), and the other part was resuspended with only 3% BSA and placed at room temperature. Incubate for 30 minutes; (6) After incubation, centrifuge at room temperature for 5 minutes, discard the supernatant, and wash the cells with 3% BSA solution; (7) Centrifuge at room temperature for 5 minutes, discard the supernatant, and incubate with 1:100 FITC-labeled anti-mouse secondary antibody prepared with 3% BSA at room temperature for 30 minutes in the dark; (8) After incubation, centrifuge at room temperature for 5 minutes, discard the supernatant, and wash the cells with PBS solution without calcium and magnesium; (9) Repeat the above steps 8 (10) Gently blow the cell pellet with PBS solution to make it a single cell to prevent cell clumps from causing damage to the experimental instrument, and then filter the cell suspension through a filter; (11) Evaluate the purity and differentiation efficiency of cardiomyocytes on a flow cytometer using standard procedures. The experimental results were analyzed using FlowJo software.

[0040] Experimental results:

[0041] like Figure 1-4 As shown, Figure 1 Add glycolysis product F-1-6-BP to the hESC-CMs cell model to replenish the experimental time nodes and flow chart; Figure 2 Immunofluorescence staining of Ki67, a proliferation marker, for the addition of glycolysis product F-1-6-BP to cell models of different groups (WT+PBS, WT+F-1,6-BP, PFKP KO+PBS, PFKP KO+F-1,6-BP). Scale bar is 50 μm; Figure 3 Quantitative analysis statistics of Ki67 immunofluorescence staining of the above four groups of models (n=5); Figure 4 Flow cytometric analysis of proliferation marker Ki67 of glycolysis product F-1-6-BP in different groups (WT+PBS, WT+F-1,6-BP, PFKP KO+PBS, PFKP KO+F-1,6-BP) of cell models (n=3). Data are expressed as mean ± standard deviation, statistical significance was determined by t-test, *P<0.05, **P<0.01, ***P<0.001, ns: no significant statistical difference.

[0042] Flow cytometry analysis showed that compared with PFKP KO cardiomyocytes, the addition of F-1,6-BP successfully increased the proportion of Ki679 (Ki67 is a cell proliferation marker, mainly used to detect the proliferation status of cells) positive (21.000±1.825vs 30.433±3.055P=0.0343), indicating that the proliferation ability of cardiomyocytes was significantly improved. The immunofluorescence results were consistent with the flow cytometry results. Compared with PFKP KO cardiomyocytes, the addition of F-1,6-BP effectively increased the proportion of Ki67 positive (20.732±3.189vs 32.908±3.731P=0.0103), indicating that the proliferation ability of cardiomyocytes was significantly improved.

[0043] In summary, F-1,6-BP can effectively rescue the decreased cardiomyocyte proliferation ability caused by PFKP gene knockout, thereby improving myocardial dysplasia.

[0044] Example 3

[0045] In vivo experiment: Effect of F-1,6-BP on embryonic myocardial development in PFKP gene knockout mice A mouse model was used to evaluate whether F-1,6-BP could restore the phenotype of embryonic myocardial dysplasia caused by PFKP deficiency.

[0046] Experimental grouping: (1) Control group (PFKP flox / flox+Saline): Serves as a negative control group for the experiment to evaluate the morphology and function of the heart during normal embryonic development. Provides a baseline data for comparing changes in other experimental groups. Any differences observed in the experimental groups can be compared with this control group to determine whether they are caused by experimental treatment (such as PFKP knockout or F-1,6-BP injection). (2) PFKP flox / flox+F-1,6-BP group: Evaluate the effect of F-1,6-BP alone on normal embryonic heart development. Determine whether F-1,6-BP has an effect on heart development in the absence of PFKP gene knockout. If there is no significant difference between this group and the control group (PFKP flox / flox+Saline), it means that F-1,6-BP itself is safe for normal embryonic heart development, and its subsequent effect in the knockout group can be attributed to compensation for PFKP deficiency. (3) PFKP cKO+Saline group: As a PFKP gene knockout model group, it is used to evaluate the effect of PFKP deficiency on embryonic heart development. It shows the phenotype of myocardial hypoplasia caused by PFKP deficiency, such as thinning of the myocardial compact layer and decreased proliferation of cardiomyocytes. This group is the main experimental group of the study, used to verify the key role of PFKP in embryonic heart development. (4) PFKP cKO+F-1,6-BP group: It is used to evaluate whether F-1,6-BP can rescue myocardial hypoplasia caused by PFKP deficiency. If the cardiac phenotype of this group (such as the thickness of the myocardial compact layer and the proliferation of cardiomyocytes) is significantly improved, it means that F-1,6-BP can restore the normal development of cardiomyocytes by supplementing the downstream metabolites of PFKP. This result will directly prove that myocardial hypoplasia caused by PFKP deficiency is due to the interruption of the glycolytic pathway, and supplementing key metabolites can be used as a potential therapeutic strategy.

[0047] At embryonic day 12.5, PFKP cKO fetal mice were injected with 500 mg / kg F-1,6-BP through the umbilical vein, and at embryonic day 17.5, the hearts of each group (PFKPflox+flox+Saline, PFKPflox+fiox+F-1,6-BP, PFKP cKO+Saline, PFKP cKO+F-1,6-BP) were evaluated for cardiac morphology and proliferation function (including immunofluorescence staining and HE staining, except for HE staining, the rest of the methods refer to Example 2).

[0048] HE staining: (1) Use a slide baking machine to bake the slides until the wax dissolves. (2) Dewax in xylene (I) for 5 minutes and dry the liquid with absorbent paper. (3) Dewax in xylene (II) for 10 minutes (the slides become transparent) and dry the liquid with absorbent paper. (4) Place in 100% ethanol for 5 minutes and dry the liquid with absorbent paper. (5) Repeat step 4. (6) Place in 95% ethanol for 3 minutes and dry the liquid with absorbent paper. (7) Place in running water for 2 minutes and dry the liquid with absorbent paper. (8) Stain with hematoxylin for 4 to 8 minutes, then rinse with tap water and dry the liquid with absorbent paper. (9) Differentiate in 1% hydrochloric acid aqueous solution for 5 to 10 seconds (the slides turn from blue to red). (10) Wash with tap water to return to blue for 15 to 30 minutes. (11) 0.5% eosin (water soluble) staining for 30 seconds to 1 minute (12) 95% ethanol (1) dehydrate for 5 minutes, and dry the liquid with absorbent paper; (13) 95% ethanol (I) for 5 minutes, and dry the liquid with absorbent paper; (14) immerse in 100% ethanol (1) for 5 minutes, and dry the liquid with absorbent paper; 16. immerse in 100% ethanol (1) for 2 minutes, and dry the liquid with absorbent paper; (15) immerse in xylene (1) for 2-3 minutes, and dry the liquid with absorbent paper; (16) immerse in xylene (Il) for 5 minutes; (17) Seal with neutral gum. (18) Microscopic observation results: cell nuclei are dark blue, and cytoplasm and fibrous tissue are red of varying shades.

[0049] Experimental results:

[0050] like Figure 5-8 As shown, Figure 5 HE-stained sections of the hearts of different groups of mice in WT+PBS, WT+F-1-6-BP, Pfkp cKO+PBS and Pfkp cKO+F-1-6-BP models at embryonic day 17.5. Scale bar = 600 μm; Figure 6 Images of Ki67, a cardiac immunofluorescence proliferation marker, in different groups of mice: WT+PBS, WT+F-1-6-BP, Pfkp cKO+PBS, and Pfkp cKO+F-1-6-BP model mice at embryonic day 17.5. Scale bar = 30 μm; Figure 7 Quantitative analysis of the thickness of the compact layer of the left and right ventricular wall in the above four groups of models (n=6); Figure 8 Quantitative analysis of Ki67 immunofluorescence staining in the above four groups of models (n=6). Data are expressed as mean ± SD, n=5 for each genotype. Statistical significance was determined by t-test *P<0.05, **P<0.01, ***P<0.001, ns: no significant statistical difference.

[0051] Cardiac morphology: Compared with PFKP conditional knockout mice (PFKP cko+Saline), early embryonic injection of F-1,6-BP (PFKP cKO+F-1,6-BP) in PFKP cKO mice could effectively alleviate the cardiac phenotype of thinning of the left ventricle (151.700±32.158vs 231.950±31.477P<0.0001) and right ventricular myocardial compact layer (121.083±18.083vs 183.2±3.731P<0.0001) in PFKP cKO mice. Figure 5 and Figure 7 ), and also alleviated the cardiac phenotype of myocardial proliferation defect in PFKP cKO mice (13.059±2.853vs 20.117±1.759P=0.000426) ( Figure 6 and Figure 8 ), which significantly increased the thickness of the myocardial compact layer of the left and right ventricles and improved the cardiac phenotype.

[0052] Myocardial cell proliferation ability: The proliferation ability of myocardial cells was detected by Ki67 immunofluorescence staining, and the results showed that the proliferation ability of myocardial cells in mice injected with F-1,6-BP was significantly improved.

[0053] In summary: F-1,6-BP can effectively alleviate embryonic myocardial dysplasia caused by PFKP gene knockout, improve cardiomyocyte proliferation ability, and alleviate the pathological phenotype of thinning of the myocardial compact layer.

[0054] Therefore, the application of F-1,6-BP in congenital heart disease drugs can make up for the shortcomings of existing technologies and provide new solutions for the prevention and treatment of embryonic myocardial dysplasia and intrauterine failure of congenital heart disease, so as to improve embryonic myocardial dysplasia and reduce the incidence of congenital heart disease and intrauterine failure of congenital heart disease.

[0055] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0056] The above description is only a specific embodiment of the present disclosure, so that those skilled in the art can understand or implement the present disclosure. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of fructose-1,6-diphosphate in the preparation of drugs for preventing or treating congenital heart disease.

2. Use of fructose-1,6-diphosphate in the preparation of drugs for preventing or treating embryonic myocardial dysplasia.

3. Use of fructose-1,6-diphosphate in the preparation of drugs for preventing or treating intrauterine heart failure in congenital heart disease.

4. Application of fructose-1,6-bisphosphate in the preparation of drugs for alleviating glycolytic dysfunction.

5. Application of fructose-1,6-diphosphate in the preparation of drugs for improving the proliferation ability of cardiomyocytes.

6. The use according to any one of claims 1 to 5, characterized in that: The medicine comprises fructose-1,6-diphosphate and a pharmaceutically acceptable excipient or carrier.

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