Application of FBP and its protective agents in the preparation of drugs that promote myocardial repair and regeneration

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of fructose-1,6-bisphosphate (FBP) and its protective agents in the preparation of drugs that promote myocardial repair and regeneration. The FBP is fructose-1,6-bisphosphate, and its structural formula is shown in Formula 1 below. The protective agent is a substance that inhibits FBP decomposition or promotes FBP synthesis. The research results of this invention show that FBP can not only promote the proliferation of cardiomyocytes after zebrafish injury and myocardial infarction in adult mice, but also directly promote the proliferation of primary cardiomyocytes in rats. More importantly, FBP injection significantly promotes the recovery of cardiac function and the reduction of fibrosis after myocardial infarction in adult rats, indicating its important promoting effect on cardiac regeneration. Formula 1.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of FBP and its protective agents in the preparation of drugs that promote myocardial repair and regeneration. Background Technology

[0002] Cardiovascular disease is the leading cause of death in humans, with ischemic cardiomyopathy, such as myocardial infarction, having a persistently high mortality rate. Acute myocardial infarction leads to myocardial ischemia and irreversible cardiomyocyte death. Due to the extremely low turnover rate of adult human cardiomyocytes, dead cardiomyocytes are gradually replaced by fibroblasts, forming permanent scars, further leading to heart failure and even death. Unlike other tissue cells, adult mammalian cardiomyocytes exit the cell cycle and become terminally differentiated multinucleated cells, thus they cannot replenish lost myocardium through proliferation after injury. However, in the neonatal period (shortly after birth), both mice and large animals like pigs exhibit strong cardiac regeneration capabilities after injury, accompanied by increased cardiomyocyte proliferation, reduced fibrosis, and recovery of cardiac function. Even in neonatal humans, there are reports of retaining the ability to repair myocardial damage and restore cardiac function. However, unlike mammals, the lower vertebrate zebrafish retains its cardiac regeneration capabilities throughout its lifespan. Through genetic pedigree tracing or isotope labeling, different research teams have elucidated that regenerated cardiomyocytes originate from the dedifferentiation and proliferation of pre-existing cardiomyocytes. Therefore, elucidating the key factors and regulatory targets that differentiate the regenerative abilities of newborn and adult mammals, as well as zebrafish, and regulating the proliferation of endogenous cardiomyocytes, has long-term and important strategic significance for the field of cardiac regeneration and the treatment of ischemic myocardial diseases.

[0003] Currently, drugs for treating ischemic myocardial disease include SGLT2 inhibitors (sodium-glucose cotransporter 2 inhibitors), ARNI (angiotensin receptor / neprilysin inhibitors), and β-blockers. When these drugs are used, they indirectly "protect" the surviving myocardium by reducing cardiac load, reducing oxygen consumption, and resisting fibrosis, thus delaying the progression to heart failure. They cannot promote cardiac regeneration by promoting the endogenous proliferation of myocardial cells. Summary of the Invention

[0004] To address the problem that existing drugs for treating ischemic myocardial disease cannot promote cardiac regeneration by stimulating endogenous proliferation of myocardial cells, this invention provides the application of FBP and its protective agents in the preparation of drugs that promote myocardial repair and regeneration. To achieve the above objective, this invention employs the following technical solution.

[0005] The first objective of this invention is to provide the application of FBP and its protective agents in the preparation of drugs that promote myocardial repair and regeneration, wherein the FBP is fructose-1,6-bisphosphate, and its structural formula is shown in Formula 1 below: Formula 1.

[0006] The protective agent is a substance that inhibits FBP decomposition or promotes FBP synthesis.

[0007] Fructose-1,6-bisphosphate (FBP) is a key metabolite in glycolysis. It is synthesized from fructose-6-phosphate via phosphofructokinase (PFK), and then further catalyzed by aldolase to produce dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P), which participate in subsequent glycolytic reactions. As a crucial metabolite, FBP has many important functions and participates in numerous physiological processes, such as antiviral activity, inhibition of inflammatory responses, activation of AMP-activated protein kinase (AMPK) in glucose-deficient conditions, and promotion of tumor cell proliferation and growth by binding to EGFR. More importantly, FBP can restore cardiac energy metabolism under hypothermic conditions, playing a protective role for the myocardium. Furthermore, intravenous injection of FBP or perfusion of FBP into an isolated heart can significantly improve myocardial function and energy metabolism after ischemia. Based on the cardioprotective effects of fibrinogen (FBP), clinical studies have explored this phenomenon. FBP administration to patients with ventricular septal defects and those who underwent heart valve surgery during cardiopulmonary bypass reduced myocardial ischemia / reperfusion injury. Furthermore, FBP administration significantly improved cardiac function in 30 patients with chronic heart failure caused by various heart diseases. These studies demonstrate the cardioprotective effects of FBP. However, to date, no research has shown whether FBP plays a significant role in cardiac regeneration, whether FBP treatment can directly promote in situ proliferation of cardiomyocytes and cardiac regeneration after myocardial infarction, whether it can alter cardiomyocyte proliferation and cardiac regeneration through endogenous regulation of FBP synthesis and degradation, or whether it can serve as a direct treatment to promote myocardial regeneration after acute myocardial infarction. Based on this, the present invention provides a small molecule metabolite, FBP, that may determine the heart's regenerative capacity. Its level in newborn mice and zebrafish with heart regeneration capacity is much higher than that in young and adult mice that cannot regenerate. Furthermore, studies have shown that it can promote the proliferation of cardiomyocytes after zebrafish injury and after myocardial infarction in adult mice, and can also directly promote the proliferation of primary cardiomyocytes in rats. Thus, the present invention provides the application of FBP and its protective agents in the preparation of drugs that promote myocardial repair and regeneration.

[0008] Existing treatments for ischemic myocardial disease (such as SGLT2 inhibitors, ARNIs, and β-blockers) indirectly "protect" surviving myocardium to delay the progression of heart failure by reducing cardiac load, decreasing oxygen consumption, and combating fibrosis. Their essence is functional support and pathological intervention for existing myocardium; they cannot achieve cardiac regeneration through endogenous promotion of cardiomyocyte proliferation, meaning they cannot induce the replenishment and tissue regeneration of damaged or lost cardiomyocytes. The 1,6-fructose diphosphate (FBP) and its protective agent used in this invention do not act through the aforementioned indirect protective mechanism, but rather promote cardiomyocyte division and proliferation from the source, achieving functional cardiac regeneration and damage repair. Therefore, this invention effectively solves the technical problem of existing drugs "only delaying heart failure but failing to achieve endogenous myocardial proliferation to drive cardiac regeneration," providing a novel regenerative medicine strategy for the treatment of ischemic myocardial disease.

[0009] Preferably, the drug is a liquid preparation.

[0010] Preferably, the effective concentration of FBP in the drug is 0.5 mM to 5 mM.

[0011] Preferably, the substance that inhibits FBP degradation is an enzyme gene that inhibits FBP degradation. Aldoa The preparation.

[0012] Preferably, the gene that inhibits the degradation of FBP is an enzyme. Aldoa The formulation is targeted Aldoa The siRNA, the sequence of the positive strand of which is shown in SEQ ID NO.5 and SEQ ID NO.7.

[0013] Preferably, the gene that inhibits the degradation of FBP is an enzyme. Aldoa The formulation is a targeted drug delivered by an AAV9 adeno-associated virus vector. Aldoa shRNA.

[0014] More preferably, the inhibitor is a targeted inhibitor. Aldoa The shRNA is delivered by an AAV9 adeno-associated virus vector to form recombinant adeno-associated virus (AAV9-). Aldoa shRNA adenovirus).

[0015] Preferably, the AAV9- Aldoa shRNA adenoviruses are used to promote cardiomyocyte proliferation and / or promote cardiac fibrosis after myocardial infarction, thereby promoting the recovery of cardiac function.

[0016] Preferably, the drug is used to promote cardiomyocyte proliferation, promote cardiac regeneration, and / or repair cardiac damage.

[0017] Preferably, the drug is used to reduce the degree of myocardial fibrosis and / or restore cardiac function.

[0018] A second objective of this invention is to provide the synthase gene for the FBP. Pfkm The application of inhibitors in the preparation of disease models that inhibit cardiomyocyte proliferation and / or promote cardiac fibrosis after myocardial infarction, exacerbating the decline in cardiac function.

[0019] Preferably, the inhibitor is a targeted inhibitor. Pfkm The siRNA, the sequence of the positive strand of which is shown in SEQ ID NO.1 and SEQ ID NO.3.

[0020] Preferably, the inhibitor is a targeted inhibitor delivered by an adeno-associated virus vector. Pfkm shRNA.

[0021] More preferably, the inhibitor is a targeted inhibitor. Pfkm The shRNA is delivered by an AAV9 adeno-associated virus vector to form recombinant adeno-associated virus (AAV9-). Pfkm shRNA adenovirus).

[0022] Preferably, the AAV9- Pfkm shRNA adenoviruses are used to inhibit cardiomyocyte proliferation and / or promote cardiac fibrosis after myocardial infarction, thereby exacerbating the decline in cardiac function.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides the application of fibroblast-derived protein (FBP) and its protectants in the preparation of drugs that promote myocardial repair and regeneration. Experiments have demonstrated that in vitro culture of effective concentrations of FBP can promote the proliferation of primary cardiomyocytes. In animal models, intraperitoneal injection of FBP can promote cardiomyocyte proliferation, reduce fibrosis, and restore cardiac function in different animal models (zebrafish, mice) after cardiac injury. Simultaneously, substances that inhibit FBP degradation can maintain the effective concentration and stability of FBP in vivo, while substances that promote FBP synthesis can enhance the production level of endogenous FBP in cardiomyocytes. Both contribute to improving cardiomyocyte proliferation efficiency, accelerating the cardiac regeneration process, and promoting the recovery of cardiac function after injury. These results indicate that FBP and its protectants do not exert their effects through traditional indirect methods such as load reduction or anti-fibrosis, but rather by directly activating the endogenous proliferation mechanism of cardiomyocytes, achieving substantial cardiac regeneration and functional repair. Therefore, this invention effectively solves the technical difficulties and problems of existing drugs for treating ischemic myocardial disease that cannot promote cardiac regeneration by activating endogenous proliferation of cardiomyocytes.

[0024] 2. This invention uses zebrafish and mice as research subjects and employs metabolomics, genetics, molecular biology, biochemistry, and cell biology to comprehensively explore the functions of FBP, FBP synthase gene, and FBP degradase gene in cardiomyocyte proliferation and regeneration.

[0025] First, non-target metabolomics analysis was performed on the hearts of mice at 1 day, 15 days, and 6 weeks of age. The results showed that FBP levels in newborn mice were significantly higher than in young and adult mice. FBP levels in zebrafish and mice of different ages were detected using a kit. The results showed that FBP levels in zebrafish were similar to those in newborn mice, but significantly higher than in young and adult mice lacking cardiac regeneration capacity. These results suggest that baseline FBP levels may be a potential determinant of regenerative capacity. Intraperitoneal injection of FBP into zebrafish after cardiac injury significantly promoted the proliferation of zebrafish cardiomyocytes. Intraperitoneal injection of FBP into mice after myocardial infarction not only significantly promoted cardiomyocyte proliferation but also reduced fibrosis and restored cardiac function. Culture experiments using different concentrations of FBP in isolated primary rat cardiomyocytes showed that 0.5 mM-5 mM FBP significantly promoted the proliferation of primary rat cardiomyocytes. These results demonstrate that FBP plays an important promoting role in myocardial proliferation and cardiac regeneration.

[0026] To investigate whether FBP promotes cardiomyocyte proliferation as a metabolite or through metabolic pathways, this invention provides a method targeting the knockdown of the FBP synthase gene. Pfkm and FBP degradase gene Aldoa siRNA was found to knock down FBP synthase. Pfkm This invention significantly inhibits the proliferation of primary rat cardiomyocytes, while knockdown of the FBP-degrading enzyme Aldoa significantly promotes the proliferation of primary rat cardiomyocytes; the invention also provides a method for knocking down the FBP synthase gene in vivo. Pfkm and FBP degradase gene Aldoa AAV9 adeno-associated virus vector, metabolomics results showed knockdown Pfkm Knockdown by reducing FBP content Aldoa Increased FBP content, functional experiments showed knockdown Pfkm It significantly exacerbated fibrosis and decreased cardiac function, while knockdown... Aldoa The results not only significantly reduced fibrosis and promoted the recovery of cardiac function, but also promoted the re-entry of cardiomyocytes into the cell cycle in adult mice. These results demonstrate that FBP acts as a metabolite signaling molecule to promote cardiomyocyte proliferation and cardiac regeneration. Attached Figure Description

[0027] Figure 1The results of detecting FBP levels in the hearts of different animal models in this invention are as follows: A is a heatmap analysis of metabolomics data from the hearts of mice at different ages (1 day, 15 days, and 6 weeks), showing that the levels of metabolites in newborn mice were significantly higher or lower than those in the hearts of young and adult mice. B represents the detection of FBP content in the hearts of mice (P1, P15, W6) and zebrafish of different ages.

[0028] Figure 2 The results of this invention on the effect of FBP injection on cardiomyocyte proliferation after cardiac injury in zebrafish are as follows: A represents the EdU staining (labeling cell cycle S phase) and MF20 (labeling cardiomyocytes) immunofluorescence experiments on zebrafish heart sections injected with FBP and PBS. B represents the statistical results of the proportion of EdU-positive cardiomyocytes (proliferating cardiomyocytes) in the area near the cardiac wound in Figure A.

[0029] Figure 3 The results of this invention on the effect of FBP injection on cardiomyocyte proliferation after cardiac injury in adult mice are as follows: A is a flowchart of the FBP injection experiment in adult mice; B represents EdU staining and CTNT (labeled cardiomyocytes) immunofluorescence experiments on adult mouse heart sections injected with FBP and PBS; C represents adult mouse heart sections injected with FBP and PBS at pH 3 (labeling cell cycle M phase), CTNT immunofluorescence assay; D represents the statistical result of the proportion of EdU-positive cardiomyocytes (proliferating cardiomyocytes) in the area near the cardiac wound in B; E represents the statistical result of the proportion of pH3-positive cardiomyocytes (proliferating cardiomyocytes) in the area near the cardiac wound in C.

[0030] Figure 4 The results of this invention on the effect of FBP injection on fibrosis following cardiac injury in adult mice are as follows: A is a marson staining experiment on damaged heart sections of adult mice injected with FBP and PBS (blue marks fibrosis). B represents the statistical analysis results of the proportion of the wound midline to the left ventricle in Figure A.

[0031] Figure 5 The results of this invention on the effect of FBP injection on cardiac function in adult mice after cardiac injury include: A represents representative echocardiograms of adult mice injected with FBP and PBS before surgery and 28 days after myocardial infarction (28dpMI). B represents the statistical analysis results of cardiac function index ejection fraction (EF) measured by heart rate superposition at different time points after myocardial infarction in adult rats; C represents the statistical analysis results of the short-axis shortening rate (FS) of the cardiac function index measured by cardiac ultrasound at different time points after myocardial infarction in adult rats.

[0032] Figure 6 The results of detecting the effect of FBP treatment on the proliferation of primary rat cardiomyocytes in this invention are as follows: A represents primary cardiomyocytes treated with different concentrations of FBP and Ki67 (labeled for proliferation), CTNT immunofluorescence assay; B represents primary cardiomyocytes treated with different concentrations of FBP at pH 3 (labeled for proliferation), as shown in the CTNT immunofluorescence assay. C represents the statistical result of the proportion of Ki67-positive cardiomyocytes (proliferating cardiomyocytes) in A; D represents the statistical result of the proportion of pH3-positive cardiomyocytes (proliferating cardiomyocytes) in B.

[0033] Figure 7 The results of this invention on the effects of knockdown of FBP synthase Pfkm and FBP degradase Aldoa on the proliferation of primary rat cardiomyocytes are as follows: A is a pair Aldoa qPCR was performed on primary cardiomyocytes after siRNA knockdown to validate RNA levels. B is the correct answer. Pfkm qPCR was performed on primary cardiomyocytes after siRNA knockdown to validate RNA levels. C represents the knockdown of FBP synthase Pfkm and FBP degrading enzyme. Aldoa Primary cardiomyocytes Ki67, CTNT immunofluorescence assay; D represents knockdown of FBP synthase. Pfkm and FBP degrading enzyme Aldoa Primary cardiomyocytes pH3, CTNT immunofluorescence assay; E represents the statistical result of the proportion of Ki67-positive cardiomyocytes (proliferating cardiomyocytes) in C; F represents the statistical result of the proportion of pH3-positive cardiomyocytes (proliferating cardiomyocytes) in D.

[0034] Figure 8 For the detection of adeno-associated virus knockdown in this invention Pfkm , Aldoa The results of FBP content in the heart of mice after the disease, including: A is a schematic diagram of adeno-associated virus injection; B is for adenovirus sh- Aldoa Western blot analysis was performed on the heart of the knocked-down mouse to verify the protein level. C represents the statistical analysis of grayscale values ​​in Figure B; D represents the response to adenovirus sh- Pfkm Western blot analysis was performed on the heart of the knocked-down mouse to verify the protein level. E represents statistical analysis of the grayscale values ​​of D; F is for knocking down Pfkm Reduce and knock down at the same time Aldoa Thermographic analysis of the added metabolites (the metabolites highlighted in red are FBP).

[0035] Figure 9 For the detection of adenovirus knockdown in this invention Pfkm , Aldoa Results of the effect of cardiac injury on fibrosis in mice, including: A is knocking down Pfkm and knock down Aldoa Masson staining experiment on damaged heart sections of adult rats; B represents the statistical analysis results of the proportion of the wound midline to the left ventricle in Figure A.

[0036] Figure 10 For the detection of adenovirus knockdown in this invention Pfkm , Aldoa The effects of cardiac injury on cardiac function in mice were observed, including: A is knocking down Pfkm and knock down Aldoa Representative echocardiograms of each group of adult rats before surgery and 28 days after myocardial infarction (28 dpMI); B represents the statistical analysis results of cardiac function index ejection fraction (EF) measured by heart rate superposition at different time points after myocardial infarction in adult rats; C represents the statistical analysis results of the short-axis shortening rate (FS) of the cardiac function index measured by cardiac ultrasound at different time points after myocardial infarction in adult rats.

[0037] Figure 11 For the detection of adenovirus knockdown in this invention Aldoa The results of cardiomyocyte proliferation after cardiac injury in mice, including: A is knocking down Aldoa EdU staining and CTNT immunofluorescence assay of heart sections from adult rats; B is for knocking down Aldoa pH3 and CTNT immunofluorescence assays of heart sections from adult rats; C represents the statistical result of the proportion of EdU-positive cardiomyocytes (proliferating cardiomyocytes) in the area near the cardiac wound in A; D represents the statistical result of the proportion of pH3-positive cardiomyocytes (proliferating cardiomyocytes) in the area near the cardiac wound in B. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0039] Example 1 1. FBP levels are related to cardiac regenerative capacity.

[0040] C57BL / 6J wild-type mice of different ages (purchased from Hangzhou Hangsi Biotechnology Co., Ltd.), namely (P1 (one day after birth) (newborn mice), P15 (15 days after birth) (juvenile mice), and W6 (6 weeks after birth) (adult mice), were euthanized by cervical dislocation. The intact hearts were removed, the atria were removed, and only the ventricles were retained. The hearts were rinsed with pre-cooled PBS to remove residual blood. Six newborn mouse (P1) hearts were pooled into one replicate, two juvenile mouse (P15) hearts were pooled into one replicate, and each adult mouse (W6) heart was a replicate, thus obtaining heart samples from mice of different ages.

[0041] Non-target metabolomics analysis was performed on cardiac samples from each group (including metabolite extraction and instrumental analysis, all of which were completed by Shanghai Baiqu Biotechnology Co., Ltd.). Figure 1 Heatmap analysis of metabolites in the hearts of newborn mice that were significantly higher or lower than those in the hearts of young and adult mice (VIP≥1, p-value<0.05), with 3 biological replicates in each group. Figure 1 (A) Using an FBP content detection kit (Solarbio, BC2240), according to the kit protocol, heart tissues from mice (P1, P15, W6) and zebrafish of different ages were ground and lysed, and enzyme-containing reagents were added. The FBP content was then detected by measuring changes in NADH using a spectrophotometer. Figure 1 (B in the middle).

[0042] Metabolomics analysis showed that FBP levels in newborn mice were much higher than in young and adult mice; FBP content detection results showed that FBP content in zebrafish hearts was similar to that in newborn mice, but much higher than that in young and adult mice that do not have regenerative capacity.

[0043] As can be seen from the above, FBP levels are related to cardiac regenerative capacity.

[0044] 2. During the regeneration of the zebrafish heart, FBP promotes the proliferation of cardiomyocytes.

[0045] Adult AB wild-type zebrafish (provided by the National Zebrafish Resource Center) over 3 months of age were anesthetized with a 2% (g / mL) tricaine bath. The zebrafish were then fixed in a sponge, and the thoracic cavity was carefully opened with forceps. The heart was excised, and the tip of the ventricle, occupying 20% ​​of the entire heart, was removed. Three to six days post-surgery, the zebrafish were intraperitoneally injected with 15 µL of FBP (fructose 1,6-bisphosphate) solution (1 mM) and 3 µL of EdU (5-ethynyl-2'-deoxyuridine) solution (0.4 M) as the experimental group. Simultaneously, the control group was injected with PBS and EdU solution. Seven days after the operation (7 dpa), the zebrafish in the experimental group and the control group were euthanized, the heart was removed, the ventricle was preserved, the blood was removed, the heart was fixed with PFA, dehydrated with sucrose, embedded with OCT, and the heart was frozen sectioned (10 µm). The sections were then stained with EdU labeling of proliferating cells (green), immunofluorescence staining with MF20 antibody labeling of cardiomyocytes (red), and DAPI staining of cell nuclei (blue). Figure 2 In the image, A represents a representative slice photograph of the area near the cardiac wound in each group. Figure 2 B in Figure 2 The statistical results of the proportion of EdU-positive cardiomyocytes in the area near the cardiac wound in sample A are shown in the figure. Each point represents an individual cardiac sample.

[0046] The FBP solution (100mM) was prepared as follows: 11 mg of FBP was dissolved in 200 µL of PBS. FBP was purchased from Meilun Biotechnology Co., Ltd., catalog number MB2873. PBS: 1×PBS, pH 7.4.

[0047] The EdU solution (0.4M) was prepared by dissolving 0.063g of EdU in 625µL of DMSO. EdU was purchased from Thermo Fisher Scientific, catalog number A10044.

[0048] The results showed that FBP injection significantly increased the frequency of cardiomyocyte proliferation during zebrafish heart regeneration.

[0049] As can be seen from the above, FBP promotes the proliferation of cardiomyocytes during the regeneration of zebrafish hearts.

[0050] 3. FBP injection significantly increased the proportion of cardiomyocytes entering the cell cycle after myocardial infarction in adult rats.

[0051] Eight-week-old wild-type C57 adult rats were anesthetized and intubated, followed by thoracic ligation of the left anterior descending coronary artery. From day 1 to day 7 post-surgery, the experimental group received daily intraperitoneal injections of FBP solution (1 mM, 1 mg / kg) and EdU solution, while the control group received an equal concentration of PBS once daily. From day 7 to day 28, FBP solution was injected twice weekly. Figure 3(A in the diagram represents the FBP injection experiment flowchart for adult mice). Seven days post-surgery (7 dpMI), heart samples were collected from mice in both the experimental and control groups. Heart samples were frozen sections stained with EdU labeling for proliferating cells (entering S phase) (green). Figure 3 Immunofluorescence staining with antibody against phosphorylated histone 3 (pH3) in phase B or during the mitotic phase (M phase) (green) Figure 3 The staining included C), myocardial cell marker cTNT antibody immunofluorescence staining (red), and DAPI cell nuclear staining (blue). Figure 3 B and Figure 3 C in the image represents a representative slice photograph of the area near the cardiac wound in each group. Figure 3 D in Figure 3 The E in the series are as follows: Figure 3 B in Figure 3 The statistical results of the proportion of EdU or pH3 positive cardiomyocytes in the area near the cardiac wound in C are shown, with each point representing each independent cardiac sample.

[0052] The results showed that FBP injection significantly increased the entry of cardiomyocytes into the cell cycle in the damaged area of ​​adult mice after myocardial infarction.

[0053] As can be seen from the above, FBP injection significantly increases the proportion of cardiomyocytes entering the cell cycle after myocardial infarction in adult rats.

[0054] 4. FBP injection significantly reduced fibrosis in the damaged area after myocardial infarction in adult rats.

[0055] FBP injection experiment as follows Figure 3 As shown in A in the figure. Serial sections of the damaged heart from adult mice 28 days after myocardial infarction (28 dpMI) were stained with Masson's solution. Figure 4 In the image, A represents slices of the infarcted region taken from left to right along the apex of the heart, spaced 200 μm apart. The bottom image shows a representative heart from the control group (PBS), while the top image shows a representative heart from the FBP injection group. Figure 4 In the figure, B represents the statistical analysis results of the proportion of the wound midline to the left ventricle, and each point represents an independent heart sample.

[0056] The results showed that FBP injection significantly reduced fibrosis in the damaged areas of adult mice with myocardial infarction.

[0057] As shown above, FBP injection significantly reduces fibrosis in the damaged area after myocardial infarction in adult rats.

[0058] 5. FBP injection significantly promotes the recovery of cardiac function after myocardial infarction in adult rats.

[0059] FBP injection experiment as follows Figure 5As shown in A in the figure. Before and after surgery, mice injected with FBP and PBS were examined using a portable digital ultrasound diagnostic instrument and a dedicated high-frequency ultrasound probe. Cardiac function was assessed via short-axis view of the sternum. Multiple continuous and stable cardiac cycle images were captured during the examination. Changes in cardiac function indicators such as ejection fraction (EF) and left ventricular fraction of shortening (FS) were analyzed and recorded using the built-in system of the ultrasound instrument. Figure 5 In the figure, A represents representative echocardiograms from each group of adult mice before surgery and 28 days after myocardial infarction (28dpMI). Figure 5 B in Figure 5 In the figure, C represents the cardiac function index ejection fraction (EF) and fractional shortening (FS) measured by cardiac ultrasound at different time points after myocardial infarction in adult rats. Gray represents the PBS group and red represents the FBP group.

[0060] The results showed that mice injected with FBP had significantly higher ejection fraction (EF) and fractional shortening (FS) from 7 to 28 days post-surgery (dpMI) than control mice injected with PBS, demonstrating that FBP injection can promote the recovery of cardiac function in adult mice after myocardial infarction. As can be seen from the above, FBP injection significantly promotes the recovery of cardiac function after myocardial infarction in adult rats.

[0061] 6. FBP significantly increased the proliferation rate of primary cardiomyocytes in rats.

[0062] Hearts were collected from 2-3 day old newborn SD wild-type rats (purchased from Hangzhou Hangsi Biotechnology Co., Ltd.). The tissue was minced and digested multiple times with collagenase II (1 mg / ml) and trypsin. The digested cell suspension was passed through 100µm and 60µm cell sieves to remove undigested tissue or cell clusters. Cells were then centrifuged and resuspended in complete culture medium (10% (g / mL) FBS solution, DMEM). After differential adhesion for 2 hours, fibroblasts were removed. Cells were then seeded into plates according to cell number. 36 hours after primary cardiomyocytes were seeded, different concentrations (0.5mM-10mM) of FBP solution were added to serum-free DMEM as experimental groups, while the control group received the same concentration of PBS. Primary cells in each group were cultured for 24 hours. Figure 6 A in Figure 6 (B) Cells were immunofluorescence stained using cell smears to analyze the proliferation of Ki67 cells. Figure 6 A in the middle), pH3 labeled staining ( Figure 6 B (green), CTNT antibody immunofluorescence staining for cardiomyocytes (red), and DAPI nuclear staining (blue). Figure 6 A in Figure 6 In the image, B represents a representative photograph of the immunofluorescence results for each group of cells. Figure 6 C in Figure 6The D in the series are respectively: Figure 6 A and Figure 6 Statistical results of the proportion of Ki67 or pH3 positive cardiomyocytes in B.

[0063] The results showed that treatment with 0.5mM to 5mM FBP significantly promoted the proliferation of primary rat cardiomyocytes.

[0064] As can be seen from the above, FBP significantly increases the proliferation rate of primary cardiomyocytes in rats.

[0065] 7. siRNA knockdown of FBP synthase Pfkm Significantly inhibits the proliferation of primary rat cardiomyocytes and knocks down FBP-degrading enzyme. Aldoa It significantly promotes the proliferation of primary rat cardiomyocytes.

[0066] The following is synthesized using sequence information: Pfkm ( Pfkm (The Ensembl database ID for the gene is ENSRNOG00000057988) siRNA: Pfkm -siRNA1 sense (sense chain): 5'-GUGCCAACAUAACCAAGUATT-3' (SEQ ID NO.1).

[0067] Pfkm -siRNA1 antisense: 5'-UACUUGGUUAUGUUGGCACTT-3' (SEQ ID NO.2).

[0068] Pfkm -siRNA2 sense (sense chain): 5'-CGCCACGGUUUCCAAUAAUT-3' (SEQ ID NO.3).

[0069] Pfkm -siRNA2 antisense: 5'-AUUAUUGGAAACCGUGGCGTT-3' (SEQ ID NO.4).

[0070] And synthesized through sequence information as follows Aldoa ( Aldoa (The Ensembl database ID for the gene is ENSRNOG00000052802) siRNA: Aldoa -siRNA1 sense (sense chain): 5'-GGGUGUAGUGCCCCUGGCUTT-3' (SEQ ID NO.5).

[0071] Aldoa -siRNA1 antisense: 5'-AGCCAGGGGCACUACACCCTT-3' (SEQ ID NO. 6).

[0072] Aldoa -siRNA2 sense (sense chain): 5'-GGGAGCAUACUCCCUCGUCTT-3' (SEQ ID NO.7).

[0073] Aldoa -siRNA2 antisense: 5'-GACGAGGGAGUAUGCUCCCTT-3' (SEQ ID NO. 8).

[0074] Rat primary cardiomyocytes were seeded in DMEM for 36 hours and then subjected to separate processes. Pfkm siRNA (details are shown above). Aldoa Transfection with siRNA (details as shown above) and NC-siRNA (details as shown above for each siRNA) was performed. After 5 hours of transfection, the cells were replaced with 10% (g / mL) FBS (fetal bovine serum) in DMEM for further culture. After 24 hours of culture, the cells were harvested, RNA was extracted, and the RNA knockdown efficiency was detected. Figure 7 A in Figure 7 (B in the text), and simultaneously perform immunofluorescence staining experiments on the smears to detect Ki67 proliferating cells ( Figure 7 C in the pH3-labeled staining Figure 7 D (green), CTNT antibody immunofluorescence staining for cardiomyocytes (red), and DAPI nuclear staining (blue). Figure 7 C in Figure 7 In the image, D represents a representative photograph of the immunofluorescence results for each group of cells. Figure 7 E and Figure 7 The F in the series are as follows: Figure 7 C and Figure 7 Statistical results of the proportion of Ki67 or pH3 positive cardiomyocytes in D.

[0075] The results showed that Pfkm as well as Aldoa Both siRNAs significantly knocked down their corresponding RNA levels, knocking down Pfkm Significantly inhibited the proliferation of primary rat cardiomyocytes, while knockdown Aldoa It significantly promoted the proliferation of primary rat cardiomyocytes.

[0076] As can be seen from the above, siRNA knocks down FBP synthase. PfkmSignificantly inhibits the proliferation of primary rat cardiomyocytes and knocks down FBP-degrading enzyme. Aldoa It significantly promotes the proliferation of primary rat cardiomyocytes.

[0077] 8. Knockdown of cardiac troponin AAV9 in adult mice using AAV9 adeno-associated virus. Pfkm Reduce cardiac FBP levels, and knock down Aldoa Increases the level of FBP in the heart.

[0078] According to mice Aldoa Gene (its Ensembl database ID is ENSMUSG00000030695) and Pfkm shRNAs were designed based on the gene information (its Ensembl database ID is ENSMUSG00000033065). The designed shRNAs are as follows:

[0079] Aldoa shRNA: 5'-GCATCCATCAACCTCAATGCT-3' (SEQ ID NO. 9).

[0080] Pfkm shRNA: 5'-GCTATGGATGAGAAGAGATTT-3' (SEQ ID NO. 10).

[0081] Will Aldoa shRNA and Pfkm The shRNA sequences were cloned into the AAV9 plasmid (pAAV-U6-EGFP), and recombinant adeno-associated virus (including AAV9-) was synthesized. Pfkm shRNA and AAV9- Aldoa shRNA). Figure 8 In this context, A represents adult mouse AAV9- Pfkm shRNA, AAV9- Aldoa Flowchart of shRNA injection experiment. Two weeks prior to myocardial infarction surgery, adult mice were injected intravenously with adenovirus (200 μL per animal per injection, dose 2 × 10⁻⁶). 12 vg / ml). Hearts were harvested 28 days post-surgery, and proteins were extracted to detect Pfkm and Aldoa protein levels. Figure 8 B and Figure 8 (D in the middle) Figure 8 C and Figure 8 In the figure, E represents the grayscale value statistical analysis after Actin protein normalization. Simultaneously, non-target metabolomics analysis was performed on cardiac samples to detect the effects of knockdown. Pfkm Reduce and knock down at the same time Aldoa Subsequent metabolites were added, and heatmap analysis was performed on these metabolites. Figure 8 (F in the original text). The AAV9 plasmid is the AAV9 adeno-associated virus vector, or simply AAV9 adeno-associated virus. This vector uses pAAV-U6-sgRNA-CMV-GFP (Addgene #85451) as its backbone, replacing the sgRNA expression cassette with an shRNA expression cassette (containing the U6 promoter, shRNA coding sequence, and termination signal), while retaining the CMV-EGFP reporter gene expression cassette, ultimately constructing the pAAV-U6-shRNA-EGFP vector. Plasmid construction and AAV9 virus packaging were both completed by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0082] The results showed that AAV9- Aldoa shRNA injection significantly reduced Aldoa protein levels in the heart, AAV9- Pfkm shRNA injection also significantly reduced Pfkm protein levels in the heart and knocked down [the protein]. Pfkm It reduced the amount of FBP in the heart, while knocking down Aldoa It increased the level of FBP in the heart.

[0083] As shown above, knocking down the cardiac function of AAV9 in adult mice using AAV9 adeno-associated virus is effective. Pfkm Reduce cardiac FBP levels, and knock down Aldoa Increases the level of FBP in the heart.

[0084] 9. Knockdown in adult mice Pfkm Significantly exacerbated fibrosis, while knockdown Aldoa It significantly reduced fibrosis.

[0085] AAV9- Pfkm shRNA and AAV9- Aldoa shRNA injection and experimental sample collection as follows Figure 9 As shown in A in the figure. Twenty-eight days after myocardial infarction in adult rats (28 dpMI), sections of the damaged heart were stained with Masson's solution. Figure 9 In the image, 'A' represents slices taken from left to right along the apex of the heart from different sections of the infarcted region, spaced 200 μm apart. The top image shows a representative heart of the control group with the virus, and the middle image shows AAV9- Aldoa Representative hearts from the shRNA injection group; the image below shows AAV9- Pfkm Representative hearts from the shRNA injection group. Figure 9 In the figure, B represents the statistical analysis results of the proportion of the wound midline to the left ventricle, and each point represents an individual heart sample.

[0086] The results showed that knocking down in the heart Pfkm It significantly exacerbated the degree of fibrosis after myocardial infarction, while knockdown... AldoaIt significantly reduced the degree of fibrosis.

[0087] As shown above, knocking down [the virus] in adult mice... Pfkm Significantly exacerbated fibrosis, while knockdown Aldoa It significantly reduced fibrosis.

[0088] 10. Knockdown in adult mice Pfkm It significantly exacerbated the decline in cardiac function, and knockdown... Aldoa It significantly promoted the recovery of heart function.

[0089] AAV9- Pfkm shRNA and AAV9- Aldoa shRNA injection and experimental sample collection as follows Figure 10 As shown in A in the diagram. Figure 10 In the image, "A" represents representative echocardiograms of adult mice before surgery (left side) and 28 days after myocardial infarction (28 dpMI) (right side). The top image shows a representative heart of the control group infected with the virus, and the middle image shows AAV9-. Aldoa Representative hearts from the shRNA injection group; the image below shows AAV9- Pfkm Representative hearts from the shRNA injection group. Figure 10 B and Figure 10 In the graph, C represents the cardiac function index ejection fraction (EF) and fractional shortening (FS) measured by cardiac echocardiography at different time points after myocardial infarction in adult mice. Gray represents the control group (virus group), and red represents AAV9- Aldoa Group, blue represents AAV9- Pfkm shRNA.

[0090] The results showed that knocking down in the heart Pfkm It significantly exacerbated the decline in cardiac function after myocardial infarction, while knockdown... Aldoa It significantly promoted the recovery of heart function.

[0091] As shown above, knocking down [the virus] in adult mice... Pfkm It significantly exacerbated the decline in cardiac function, and knockdown... Aldoa It significantly promoted the recovery of heart function.

[0092] 11. Knockdown in adult mice Aldoa It significantly promotes the proliferation of cardiomyocytes after myocardial infarction.

[0093] AAV9- Pfkm shRNA and AAV9- Aldoa shRNA injection and experimental sample collection as follows Figure 11 As shown in A. Seven days post-surgery (7 dpMI), heart samples were frozen sectioned for EdU-labeled staining of proliferating cells (entering S phase) (green). Figure 11 Immunofluorescence staining with antibody against phosphorylated histone 3 (pH3) in phase A or during the mitotic phase (M phase) (green) Figure 11 (B) Immunofluorescence staining with cTNT antibody (red) for cardiomyocytes, and DAPI staining of cell nuclei (blue). Figure 11 A and Figure 11 B in the image represents a representative slice photograph of the area near the cardiac wound in each group. Figure 11 C and Figure 11 The D in the series are respectively: Figure 11 A and Figure 11 The statistical results of the proportion of EdU or pH3 positive cardiomyocytes in the area near the cardiac wound in sample B, with each point representing each independent cardiac sample.

[0094] The results showed that knocking down Aldoa It significantly promoted the proliferation of cardiomyocytes after myocardial infarction in adult mice.

[0095] As shown above, knocking down [the virus] in adult mice... Aldoa It significantly promotes the proliferation of cardiomyocytes after myocardial infarction.

[0096] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0097] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. The application of FBP and its protective agents in the preparation of drugs that promote myocardial repair and regeneration, characterized in that, The FBP is fructose-1,6-bisphosphate, and its structural formula is shown in Formula 1 below: Formula 1; The protective agent is a substance that inhibits FBP decomposition or promotes FBP synthesis.

2. The application according to claim 1, characterized in that, The drug is a liquid preparation.

3. The application according to claim 2, characterized in that, The concentration of FBP in the drug is 0.5 mM to 5 mM.

4. The application according to claim 1, characterized in that, The substance that inhibits FBP degradation is an enzyme gene that inhibits FBP degradation. Aldoa The preparation.

5. The application according to claim 4, characterized in that, The enzyme gene that inhibits the degradation of FBP Aldoa The formulation is targeted Aldoa The siRNA, the sequence of the positive strand of which is shown in SEQ ID NO.5 and SEQ ID NO.

7.

6. The application according to claim 4, characterized in that, The enzyme gene that inhibits the degradation of FBP Aldoa The formulation is a targeted drug delivered by an AAV9 adeno-associated virus vector. Aldoa shRNA.

7. The application according to any one of claims 1 to 6, characterized in that, The drug is used to promote cardiomyocyte proliferation, promote cardiac regeneration, and / or repair cardiac damage.

8. The application according to any one of claims 1 to 6, characterized in that, The drug is used to reduce the degree of myocardial fibrosis and / or restore cardiac function.

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