Application of coenzyme A in preparation of myocardial cell ferroptosis inhibitor

Abstract

The invention relates to the field of biological medicine, and discloses application of coenzyme A in preparation of a myocardial cell ferroptosis inhibitor. The coenzyme A has the function of inhibiting myocardial cell ferroptosis for the first time, and the function is realized by combining IGFBP2 protein, so that a new drug is expected to be developed for targeted inhibition of myocardial cell ferroptosis, and a new theoretical basis and a potential intervention strategy are provided for myocardial injury repair.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of coenzyme A in the preparation of ferroptosis inhibitors for cardiomyocytes. Background Art

[0002] Ferroptosis is a newly discovered type of programmed cell death different from apoptosis, necrosis, pyroptosis, and autophagy in recent years. It is closely related to cell metabolic disorders, redox imbalance, etc. Its main hallmark is the deposition of iron-dependent lipid peroxides in cells, and morphologically, the mitochondria in cells show a reduced volume, increased membrane density, and fewer cristae.

[0003] Currently, a large number of studies have shown that ferroptosis of cardiomyocytes is closely related to the occurrence and development of various heart diseases, including drug-induced heart failure, myocardial ischemia-reperfusion injury, septic cardiomyopathy, arrhythmia, and diabetic cardiomyopathy. In mouse models of ischemia-reperfusion injury and doxorubicin-induced cardiomyopathy, obvious ferroptosis characteristics of cardiomyocytes appear in the heart tissue. Intervention with ferroptosis inhibitors can significantly improve the pathological damage of the mouse heart and increase the survival rate of mice. Therefore, targeted inhibition of ferroptosis of cardiomyocytes is a potentially effective means for treating heart diseases.

[0004] Coenzyme A is a coenzyme containing adenosine diphosphate and derivatives of pantothenic acid (vitamin B5), which plays a key role in various metabolic reactions in the body. PANK1 is the first key enzyme for the synthesis of coenzyme A. The main function of coenzyme A is to combine with enzymes to promote the synthesis and decomposition of fatty acids, especially to participate in the β-oxidation process of fatty acids, which is one of the important ways for the body to supply energy. At the same time, coenzyme A also participates in gluconeogenesis, cholesterol synthesis, and the generation of other important biomolecules. Coenzyme A has been reported to be used for the adjuvant treatment of chronic diseases such as nephrotic syndrome, uremia (CN105407899A), chronic heparin, etc. However, there is currently no research report on the effect of coenzyme A on ferroptosis of cardiomyocytes, and the downstream molecular mechanism is not yet clear.

[0005] Insulin-like growth factor-binding protein 2 (IGFBP2) is one of the members of the IGFBP family. Proteins in this family mainly participate in regulating the biological activity of insulin-like growth factor (IGF). IGFBP2 plays an important role in various physiological and pathological processes, including cell proliferation, differentiation, migration, and apoptosis. However, there is currently no research report on the effect of IGFBP2 on ferroptosis of cardiomyocytes. Summary of the Invention

[0006] The present invention provides the use of coenzyme A in the preparation of an inhibitor of ferroptosis in cardiomyocytes. The present invention discloses for the first time that coenzyme A has the function of inhibiting ferroptosis in cardiomyocytes, and it is found that this function is achieved by binding to the IGFBP2 protein. Therefore, it is expected to develop new drugs for targeted inhibition of ferroptosis in cardiomyocytes, providing a new theoretical basis and potential intervention strategies for myocardial injury repair.

[0007] The specific technical solution of the present invention is as follows: In the first aspect, the present invention provides the use of coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in the preparation of an inhibitor of ferroptosis in cardiomyocytes.

[0008] The present invention discovers for the first time that coenzyme A has the function of inhibiting ferroptosis in cardiomyocytes. Specifically, the present invention discovers through experiments that coenzyme A can reduce the accumulation of lipid peroxides in cardiomyocytes by binding to IGFBP2, thereby inhibiting ferroptosis in cardiomyocytes. Therefore, coenzyme A can be used to treat diseases related to ferroptosis in cardiomyocytes.

[0009] Similarly, a reagent that can promote the synthesis of coenzyme A in cells can overexpress the related genes for synthesizing coenzyme A, thereby promoting the synthesis of coenzyme A in cells and increasing the content of coenzyme A, thus achieving an effect similar to that of exogenous intake of coenzyme A. And an IGFBP2 inhibitor can inhibit the expression of IGFBP2 or inhibit the function of IGFBP2, thus achieving an effect similar to that of exogenous intake of coenzyme A.

[0010] In the second aspect, the present invention provides the use of coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in the preparation of a tool drug with the function of inhibiting ferroptosis in cardiomyocytes. The tool drug can be used for scientific research. For example, the function of coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in inhibiting ferroptosis in cardiomyocytes can be used to construct relevant in vitro cell models in in vitro experiments.

[0011] In the third aspect, the present invention provides the use of coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in the preparation of a therapeutic drug for diseases related to ferroptosis in cardiomyocytes.

[0012] Furthermore, the diseases related to ferroptosis in cardiomyocytes include drug-induced heart failure, myocardial ischemia-reperfusion injury, septic cardiomyopathy, diabetic cardiomyopathy, etc.

[0013] Preferably, the therapeutic drug includes at least one of coenzyme A, a reagent that can promote the synthesis of coenzyme A in cells, and an IGFBP2 inhibitor, as well as a pharmaceutically acceptable carrier, excipient, or solvent.

[0014] More preferably, the therapeutic drug is an oral preparation or an injection preparation.

[0015] In a fourth aspect, the present invention provides the use of a drug containing coenzyme A and / or a reagent that can promote the synthesis of coenzyme A intracellularly and / or an IGFBP2 inhibitor in inhibiting ferroptosis of cardiomyocytes in vitro: Coenzyme A inhibits ferroptosis of cardiomyocytes in vitro by binding to IGFBP2, and the IGFBP2 inhibitor inhibits ferroptosis of cardiomyocytes in vitro by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

[0016] In a fifth aspect, the present invention provides a method for inhibiting ferroptosis of cardiomyocytes under in vitro conditions: adding a drug containing coenzyme A and / or a reagent that can promote the synthesis of coenzyme A intracellularly and / or an IGFBP2 inhibitor to the culture system of cardiomyocytes in vitro, and coenzyme A inhibits ferroptosis of cardiomyocytes in vitro by binding to IGFBP2. Specifically, the coenzyme A inhibits ferroptosis of cardiomyocytes by binding to IGFBP2 and reducing the accumulation of lipid peroxides in cardiomyocytes induced by cystine deprivation; while the IGFBP2 inhibitor inhibits ferroptosis of cardiomyocytes in vitro by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

[0017] Preferably, the reagent that can promote the synthesis of coenzyme A intracellularly is an adenovirus vector targeting cardiomyocytes inserted with the PANK1 gene sequence; more preferably, it is PZ-2891.

[0018] Preferably, the IGFBP2 inhibitor is an IGFBP2 neutralizing antibody, small interfering RNA (siRNA) against IGFBP2, or antisense oligonucleotide (ASO). Among them, the IGFBP2 neutralizing antibody can block its interaction with insulin-like growth factor (IGF) or other ligands by binding to IGFBP2, and inhibit the function of IGFBP2. Small interfering RNA (siRNA) or antisense oligonucleotide (ASO) against IGFBP2 can bind to IGFBP2 mRNA and inhibit the expression of IGFBP2.

[0019] Preferably, the drug includes at least one of coenzyme A, a reagent that can promote the synthesis of coenzyme A intracellularly, and an IGFBP2 inhibitor, as well as a pharmaceutically acceptable carrier, excipient, or solvent.

[0020] Preferably, the effective concentration of coenzyme A in the in vitro cardiomyocyte culture system is 50 - 500 μM.

[0021] Sixth aspect, the present invention provides a method for constructing an in vitro model of cardiomyocytes with low lipid peroxide accumulation: constructing a culture system for in vitro cardiomyocytes deprived of cystine, and simultaneously adding a drug containing coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or a drug of an IGFBP2 inhibitor (when the IGFBP2 inhibitor is a small interfering RNA or antisense oligonucleotide against IGFBP2, transfection needs to be carried out in advance). Coenzyme A reduces the accumulation of lipid peroxides in cardiomyocytes by binding to IGFBP2, thereby obtaining an in vitro model of cardiomyocytes with low lipid peroxide accumulation; the IGFBP2 inhibitor reduces the accumulation of lipid peroxides in cardiomyocytes by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2, thereby obtaining an in vitro model of cardiomyocytes with low lipid peroxide accumulation.

[0022] Seventh aspect, the present invention provides a method for constructing an in vitro model of cardiomyocytes with low lipid peroxide accumulation: first knocking out or knocking down the IGFBP2 gene of in vitro cardiomyocytes, and then culturing the in vitro cardiomyocytes in a cystine-free culture system, thereby obtaining an in vitro model of cardiomyocytes with low lipid peroxide accumulation.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention first proposes that coenzyme A can reduce ferroptosis of cardiomyocytes by binding to IGFBP2, provides the application of coenzyme A in the preparation of inhibitors of cardiomyocyte ferroptosis, and further provides the application in the preparation of drugs for treating diseases related to cardiomyocyte ferroptosis. It is expected to develop new drugs targeting cardiomyocyte ferroptosis and provide a new theoretical basis and potential intervention strategy for myocardial injury repair. Description of the Drawings

[0024] Figure 1 For primary neonatal mouse cardiomyocytes, they were cultured in complete medium for 24 hours, and then changed to complete medium [Cystine (+)] or cystine-free medium [Cystine (-)], cystine-free medium containing 500 μM coenzyme A [Cystine (-)+CoA], and cystine-free medium containing 1 μM Ferrostatin-1 (Fer-1, ferroptosis inhibitor) as a positive control. After 48 hours, the cell morphology was observed under a light microscope, scale bar = 100 μm.

[0025] Figure 2They were primary neonatal mouse cardiomyocytes, cultured in complete medium for 24 hours, and then changed to complete medium [Cystine (+)] or cystine-free medium [Cystine (-)], cystine-free medium containing 500 μM coenzyme A [Cystine (-)+CoA], and cystine-free medium containing 1 μM Ferrostatin-1 (Fer-1, ferroptosis inhibitor) [Cystine (-)+Fer-1] as a positive control. After 48 hours, cell viability was detected to evaluate the survival of cardiomyocytes, and the statistical results of the survival of cardiomyocytes in the four groups. **P<0.01, ***P<0.001, ****P<0.0001.

[0026] Figure 3 They were primary neonatal mouse cardiomyocytes, cultured in complete medium for 24 hours, and then changed to complete medium [Cystine (+)] or cystine-free medium [Cystine (-)], cystine-free medium containing 500 μM coenzyme A [Cystine (-)+CoA]. After 36 hours, cell proteins were collected, and the expression of 4HNE was detected by Western Blot to evaluate the effect of coenzyme A on lipid peroxidation. The statistical results of 4HNE expression in cardiomyocytes in the three groups (n = 6), *P<0.05, ***P<0.001.

[0027] Figure 4 They were primary neonatal mouse cardiomyocytes, cultured in complete medium for 24 hours and then changed to complete medium [Cystine (+)] or cystine-free medium [Cystine (-)], cystine-free medium containing 500 μM coenzyme A [Cystine (-)+CoA], and cystine-free medium containing 1 μM Ferrostatin-1 (Fer-1, ferroptosis inhibitor) [Cystine (-)+Fer-1] as a positive control. After 48 hours, they were stained with BODIPY 581 / 591 C11 (Invitrogen, D3861), and the effect of coenzyme A on lipid peroxidation was evaluated by flow cytometry. ****P<0.0001.

[0028] Figure 5 For C57BL / 6 mice, an adeno-associated virus overexpressing PANK1 with a cardiomyocyte-specific promoter was injected into the tail vein. Four weeks later, a model of cardiac ischemia-reperfusion injury was established. Before modeling (baseline) and 1 day, 3 days, 7 days, and 28 days after modeling, a small animal cardiac ultrasound was used to evaluate the effect of PANK1 overexpression on cardiac function in mice with cardiac ischemia-reperfusion injury. *P<0.05, **P<0.01.

[0029] Figure 6 To use Lip-MS technology to screen for target proteins in cardiomyocytes that may bind to coenzyme A.

[0030] Figure 7 Primary neonatal mouse cardiomyocytes were transfected with IGFBP2 siRNA or control reagent for 48 hours, then changed to complete medium [Cystine (+)] or cystine-free medium [Cystine (-)], cystine-free medium containing 500 μM coenzyme A [Cystine (-)+CoA]. After 48 hours, cell viability was detected to evaluate the survival of cardiomyocytes, and the statistical results of the survival of the four groups of cardiomyocytes are shown. **P<0.01, ***P<0.001, ****P<0.0001.

[0031] Figure 8 Primary neonatal mouse cardiomyocytes were transfected with IGFBP2 siRNA or control reagent for 48 hours, then changed to complete medium [Cystine (+)] or cystine-free medium [Cystine (-)], cystine-free medium containing 500 μM coenzyme A [Cystine (-)+CoA]. After 48 hours, cell proteins were collected, and the expressions of IGFBP2 and 4HNE were detected by Western Blot to evaluate the effect of knocking out IGFBP2 on lipid peroxidation in cardiomyocytes. Specific embodiments

[0032] The present invention will be further described below in conjunction with embodiments.

[0033] General embodiment In a first aspect, the present invention provides the use of coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in the preparation of an inhibitor of ferroptosis in cardiomyocytes.

[0034] In a second aspect, the present invention provides the use of coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in the preparation of a tool drug with the function of inhibiting ferroptosis in cardiomyocytes.

[0035] In a third aspect, the present invention provides the use of coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in the preparation of a therapeutic drug for diseases related to ferroptosis in cardiomyocytes.

[0036] Furthermore, the diseases related to ferroptosis in cardiomyocytes include drug-induced heart failure, myocardial ischemia-reperfusion injury, septic cardiomyopathy, diabetic cardiomyopathy, etc.

[0037] In some preferred embodiments, the therapeutic drug comprises at least one of coenzyme A, a reagent that can promote the synthesis of coenzyme A in cells, and an IGFBP2 inhibitor, as well as a pharmaceutically acceptable carrier, excipient or solvent.

[0038] In some more preferred embodiments, the therapeutic drug is an oral preparation or an injection preparation.

[0039] Fourthly, the present invention provides the use of a drug containing coenzyme A or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in inhibiting ferroptosis of cardiomyocytes in vitro: the coenzyme A inhibits ferroptosis of cardiomyocytes in vitro by binding to IGFBP2; while the IGFBP2 inhibitor inhibits ferroptosis of cardiomyocytes in vitro by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

[0040] Fifthly, the present invention provides a method for inhibiting ferroptosis of cardiomyocytes under in vitro conditions: adding a drug containing coenzyme A and / or a reagent that can promote the synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor to the culture system of cardiomyocytes in vitro, and the coenzyme A inhibits ferroptosis of cardiomyocytes in vitro by binding to IGFBP2. Specifically, the coenzyme A inhibits ferroptosis of cardiomyocytes by binding to IGFBP2 and reducing the accumulation of lipid peroxides in cardiomyocytes induced by cystine deprivation; while the IGFBP2 inhibitor inhibits ferroptosis of cardiomyocytes in vitro by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

[0041] In some preferred embodiments, the reagent that can promote the synthesis of coenzyme A in cells is an adenovirus vector targeting cardiomyocytes inserted with the PANK1 gene sequence; more preferably PZ-2891.

[0042] In some preferred embodiments, the IGFBP2 inhibitor is an IGFBP2 neutralizing antibody (more preferably from Qicheng Biotech, product number QC-5842R), small interfering RNA (siRNA) targeting IGFBP2 or antisense oligonucleotide (ASO). Among them, the IGFBP2 neutralizing antibody can block its interaction with insulin-like growth factor (IGF) or other ligands by binding to IGFBP2, and inhibit the function of IGFBP2. Small interfering RNA (siRNA) or antisense oligonucleotide (ASO) targeting IGFBP2 can bind to IGFBP2 mRNA and inhibit the expression of IGFBP2. In some preferred embodiments, the drug comprises at least one of coenzyme A, a reagent that can promote the synthesis of coenzyme A in cells, and an IGFBP2 inhibitor, as well as a pharmaceutically acceptable carrier, excipient or solvent.

[0043] In some preferred embodiments, the effective concentration of coenzyme A in the in vitro cardiomyocyte culture system is 50 - 500 μM.

[0044] In a sixth aspect, the present invention provides a method for constructing an in vitro model of cardiomyocytes with low lipid peroxide accumulation: constructing a culture system for in vitro cardiomyocytes deprived of cystine, and simultaneously adding a drug containing coenzyme A and / or a reagent that can promote the synthesis of coenzyme A intracellularly and / or an IGFBP2 inhibitor (when the IGFBP2 inhibitor is small interfering RNA or antisense oligonucleotide targeting IGFBP2, transfection needs to be carried out in advance). Coenzyme A reduces lipid peroxide accumulation in cardiomyocytes by binding to IGFBP2, thereby obtaining an in vitro model of cardiomyocytes with low lipid peroxide accumulation; the IGFBP2 inhibitor reduces lipid peroxide accumulation in cardiomyocytes by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2, thereby obtaining an in vitro model of cardiomyocytes with low lipid peroxide accumulation.

[0045] In a seventh aspect, the present invention provides a method for constructing an in vitro model of cardiomyocytes with low lipid peroxide accumulation: first knocking out or knocking down the IGFBP2 gene of in vitro cardiomyocytes, and then culturing the in vitro cardiomyocytes in a cystine-free culture system, thereby obtaining an in vitro model of cardiomyocytes with low lipid peroxide accumulation. Specific examples Example 1: Coenzyme A treatment can inhibit morphological changes of cardiomyocytes caused by cystine deficiency (1) Isolation and culture of primary cardiomyocytes from neonatal mice: Use a sterilized ophthalmic scissors to cut the hearts of 1-day-old neonatal mice. After washing once with PBS, cut into small pieces and digest with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37 °C for 10 minutes each time, for a total of 5 times. After each digestion, transfer the supernatant to a high-glucose DMEM solution containing 20% FBS for 1:1 neutralization. After digestion, filter the resulting solution containing cardiomyocytes through a 70 μm filter, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in a DMEM solution containing 10% FBS, then transfer the cells to a 10 cm cell culture dish, and perform differential adhesion in a 37 °C cell culture incubator for 90 minutes. Collect the supernatant, perform cell counting, dilute with a DMEM solution containing 10% FBS, and add Brdu at a concentration of 0.05 mM to inhibit the proliferation of fibroblasts. Subsequently, evenly seed the cardiomyocytes into a 12-well plate, and the seeding density is 5×10 5 cells / well.

[0047] (2)Cystine deprivation induces ferroptosis and coenzyme A intervention: The culture medium of primary cardiomyocytes was replaced with complete medium containing 10% FBS (normal control group), cystine-free medium (model group), cystine-free medium supplemented with coenzyme A (coenzyme A treatment group), and cystine-free medium supplemented with Fer-1 (positive treatment group), and cultured for 48 hours.

[0048] (3)Observe and record cell morphology under light microscopy.

[0049] (4)As Figure 1 shown, under the culture condition of cystine-free [Cystine (-)+DMSO], after 48 hours, compared with the normal control group [Cystine (+)+DMSO], cardiomyocytes showed obvious shrinkage, smaller volume, and cell death. After coenzyme A treatment [Cystine (-)+CoA], the morphology of cardiomyocytes was significantly improved compared with the cystine-free group [Cystine (-)+DMSO]. This indicates that coenzyme A treatment can inhibit the morphological changes of cardiomyocytes caused by cystine deficiency.

[0050] Example 2: Coenzyme A has a regulatory effect on the survival of cardiomyocytes (1)Isolation and culture of primary cardiomyocytes from neonatal mice: The hearts of neonatal mice within 1 day after birth were cut with a sterilized ophthalmic scissors, washed once with PBS, then minced, digested with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37°C for 10 minutes each time, for a total of 5 times. After each digestion, the supernatant was transferred to a high-glucose DMEM solution containing 20% FBS for 1:1 neutralization. After digestion, the solution containing cardiomyocytes was filtered through a 70μm filter, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in a high-glucose DMEM solution containing 10% FBS. Then the cells were transferred to a 10 cm cell culture dish and subjected to differential adhesion in a 37°C cell culture incubator for 90 minutes. The supernatant was collected, cell counting was performed, diluted with a high-glucose DMEM solution containing 10% FBS, and 0.05 mM Brdu was added to inhibit the proliferation of fibroblasts. Subsequently, the cardiomyocytes were evenly seeded into 96-well plates at an inoculation density of 5×10 4 cells / well.

[0051] (2)Cystine deprivation induces ferroptosis and coenzyme A intervention: The culture medium of primary cardiomyocytes was replaced with complete medium containing 10% FBS (normal control group), cystine-free medium (model group), cystine-free medium supplemented with coenzyme A (coenzyme A treatment group), and cystine-free medium supplemented with Fer-1 (positive treatment group), and cultured for 48 hours.

[0052] (3) Cell viability identification: The CCK8 kit (Beyotime, C0041) was used to evaluate the survival of neonatal rat cardiomyocytes.

[0053] (4) As Figure 2 shown, under the culture condition without cystine [Cystine (-)+DMSO], the viability of primary cardiomyocytes in the model group of neonatal rats was 7.03±1.43%, which was significantly decreased compared with that in the normal culture [Cystine (+)]. However, the addition of Fer-1 [Cystine (-)+Fer-1] could increase the survival rate of cardiomyocytes to 67.02±10.98%, inhibiting the occurrence of ferroptosis in cardiomyocytes. The treatment with coenzyme A [Cystine (-)+CoA] could increase the survival rate of cardiomyocytes to 63.15±11.40%, indicating that the treatment with coenzyme A could inhibit the occurrence of cardiomyocyte death caused by cystine deprivation, suggesting that coenzyme A has a direct regulatory effect on the survival of cardiomyocytes.

[0054] Example 3: Coenzyme A treatment can reduce the expression of 4HNE protein in cardiomyocytes (1) Isolation and culture of primary cardiomyocytes from neonatal mice: The hearts of 1-day-old neonatal mice were cut with a sterilized ophthalmic scissors, washed once with PBS, then minced, digested with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37°C for 10 minutes each time, for a total of 5 times. After each digestion, the supernatant was transferred to a DMEM solution containing 20% FBS for 1:1 neutralization. After digestion, the solution containing cardiomyocytes was filtered through a 70μm filter, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in a DMEM solution containing 10% FBS. Then the cells were transferred to a 10cm cell culture dish and differentially adhered in a 37°C cell culture incubator for 90 minutes. Subsequently, the culture dish was taken out, the supernatant was collected, cell counting was performed, diluted with a DMEM solution containing 10% FBS, and 0.05mM Brdu was added to inhibit the proliferation of fibroblasts. Then it was evenly seeded into a 12-well plate at an inoculation density of 5×10 5 cells / well.

[0055] (2) Induction of ferroptosis by cystine deprivation and intervention with coenzyme A: The culture medium of primary cardiomyocytes was changed to a complete medium containing 10% FBS (normal control group) or a cystine-free medium (model group), a cystine-free medium supplemented with coenzyme A (coenzyme A treatment group), or a cystine-free medium supplemented with Fer-1 (positive treatment group), and cultured for 48 hours.

[0056] (3)Detection of 4HNE protein expression: Discard the culture medium, wash twice with pre-cooled PBS, add RIPA lysis buffer containing phosphatase inhibitor and protease inhibitor, and lyse on ice for 30 minutes. Quantify the protein by BCA method, add 5×loading buffer, and heat the protein at 98°C in a metal bath for 30 minutes to denature it. Subsequently, perform immunoblotting experiment. The primary antibody is Anti-4 Hydroxynonenal antibody (Abcam, ab46545, 1:1000), and the secondary antibody is Goat anti-rabbit IgG H&L (Abcam, ab205718, 1:10000). Detect the expression level of 4HNE protein to evaluate the effect of coenzyme A on lipid peroxidation.

[0057] (4)As Figure 3 shown, after 48 hours of cystine deprivation, the expression of 4HNE in cardiomyocytes of the model group [Cystine (-)] was significantly higher than that of the normal control group [Cystine (+)], suggesting that the content of intracellular lipid peroxidation increased significantly after cystine deprivation. While the expression of 4HNE in the coenzyme A treatment group [Cystine (-)+CoA] was significantly lower than that of the model group, indicating that coenzyme A treatment could significantly reduce the accumulation of lipid peroxidation after cystine deprivation, and coenzyme A treatment could inhibit ferroptosis in cardiomyocytes.

[0058] Example 4: Coenzyme A treatment can inhibit lipid peroxidation in cardiomyocytes (1)Isolation and culture of primary neonatal mouse cardiomyocytes: Cut the hearts of 1-day-old neonatal mice with sterile ophthalmic scissors, wash once with PBS, then cut into pieces, and digest with 0.05% trypsin + 0.05% type II collagenase at 37°C on a shaker for 10 minutes each time, for a total of 5 times. After each digestion, transfer the supernatant to high-glucose DMEM solution containing 20% FBS for 1:1 neutralization. After digestion, filter the obtained solution containing cardiomyocytes through a 70μm filter, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in DMEM solution containing 10% FBS, then transfer the cells to a 10 cm cell culture dish, and perform differential adhesion in a 37°C cell culture incubator for 90 minutes. Collect the supernatant, count the cells, dilute with DMEM solution containing 10% FBS, and add 0.05 mM Brdu to inhibit the proliferation of fibroblasts. Subsequently, evenly seed the cardiomyocytes into 6-well plates at an inoculation density of 1×10 6 cells / well.

[0059] (2)Induction of ferroptosis by cystine deprivation and intervention with coenzyme A: Replace the culture medium of primary cardiomyocytes with complete medium containing 10% FBS (normal control group) or cystine-free medium (model group), cystine-free medium supplemented with coenzyme A (coenzyme A treatment group), and cystine-free medium supplemented with Fer-1 (positive treatment group), and culture for 48 hours.

[0060] (3) Stain with 1 μM BODIPY 581 / 591 C11 (Invitrogen, D3861), incubate at 37 °C for 30 minutes, digest with trypsin, neutralize, wash with PBS, and then evaluate the lipid peroxide content among groups using flow cytometry.

[0061] (4) As Figure 4 shown, after 48 hours of cystine deprivation, flow cytometry analysis showed that the mean fluorescence intensity of the FITC channel in cardiomyocytes of the model group [Cystine (-)] was significantly higher than that of the normal control group [Cystine (+)], indicating a significant increase in the intracellular lipid peroxide content after cystine deprivation. The mean fluorescence intensity of the FITC channel in the coenzyme A treatment group [Cystine (-)+CoA] was significantly lower than that of the model group, suggesting that coenzyme A treatment could significantly reduce the accumulation of lipid peroxides after cystine deprivation, and coenzyme A treatment could inhibit ferroptosis of cardiomyocytes.

[0062] Example 5: Overexpression of coenzyme A synthetase PANK1 can improve cardiac function in mice with cardiac ischemia-reperfusion injury (1) Tail vein injection of PANK1 overexpression adeno-associated virus (vector name GV57; element order: cTNTp-MCS-3Flag-T2A-EGFP; sequence is the CDS sequence of the gene NM_001114339.2): Select 4-week-old male C57BL / 6 mice, and inject 150 μL of saline containing 3×10 11 virus amount into each mouse through the tail vein. Mice injected with the control vector virus were set as the control group.

[0063] (2) Establish a model of cardiac ischemia-reperfusion injury in mice: Four weeks after adeno-associated virus injection, anesthetize the mice by intraperitoneal injection of chloral hydrate (100 mg / kg), intubate the trachea and connect to a small animal ventilator after anesthesia. Incise the chest wall at the 4th intercostal space on the left side of the chest to expose the heart and the left anterior descending coronary artery (LAD). Carefully pass a 6-0 silk thread through the LAD and tie a slipknot under it. When tying the slipknot, confirm that the myocardium of the anterior wall of the left ventricle turns pale, indicating successful ischemia. The ischemia time is set to 45 minutes. Then loosen the slipknot to restore coronary blood flow and complete reperfusion. After the operation, suture the chest wall and skin, and provide warming and care after the operation. In the sham operation group (Sham), the silk thread was only passed through the LAD and then immediately removed without ligation.

[0064] (3)Mouse cardiac function assessment: The mice were placed on an ultrasonic imaging platform, fixed with their backs downwards, and ultrasonic coupling agent without air bubbles was applied to the shaved area of the chest. The MS400 probe (center frequency: 30 MHz) of the VEVO 3100 system (FUJIFILM Visual Sonics, Canada) was used to obtain long-axis views and short-axis views. Under the long-axis view in M-mode ultrasonic imaging, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), etc. were recorded and analyzed. The results showed that promoting the endogenous synthesis of coenzyme A in cardiomyocytes by overexpressing PANK1 could improve the cardiac function of mice with ischemia-reperfusion injury Example 6: Mass spectrometry screening revealed that IGFBP2 is a binding target of coenzyme A (1)Isolation and culture of primary cardiomyocytes from neonatal mice: The hearts of 1-day-old neonatal mice were excised with sterilized ophthalmic scissors, washed once with PBS, then minced, and digested with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37°C for 10 minutes each time, for a total of 5 times. After each digestion, the supernatant was transferred to high-glucose DMEM solution containing 20% FBS for 1:1 neutralization. After digestion, the resulting solution containing cardiomyocytes was filtered through a 70-μm filter, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in DMEM solution containing 10% FBS. Then the cells were transferred to a 10-cm cell culture dish and subjected to differential adhesion in a 37°C cell culture incubator for 90 minutes. The supernatant was collected, cell counting was performed, diluted with DMEM solution containing 10% FBS, and 0.05 mM Brdu was added to inhibit the proliferation of fibroblasts. Subsequently, the cardiomyocytes were evenly seeded into a 10-cm cell culture dish at an inoculation density of 6×10 6 cells / dish.

[0065] (2)Cell sample preparation: After 24 hours of culture, the cells were washed twice with pre-cooled PBS, then scraped off with a cell scraper, centrifuged, and the supernatant was discarded. The cell pellet was snap-frozen in liquid nitrogen. Mass spectrometry water was added to the sample, ground at low temperature, and sonicated in an ice-water bath for 20 minutes. Centrifuged at 12000 rpm at 4°C for 10 minutes, and the supernatant was transferred to a new EP tube for BCA quantification. 600 μL of the extracted total protein was divided into 6 portions, with every 3 portions as a group. One group was added with solvent (PBS) as the control group (H1, H2, H3), and the other group was added with 33 mMol of coenzyme A as the experimental group (D1, D2, D3). PK was added at a ratio of 1:100, incubated at room temperature for 5 minutes, incubated at 95°C for 5 minutes, and the sample was allowed to cool to room temperature naturally and 2% SDC; 200 mM ABC was added. Subsequently, after the two steps of trypsin digestion and peptide elution, the sample was subjected to on-machine detection.

[0066] (3) nano-LC-MS / MS detection: 2 μL of total peptides from each sample were separated by the nano-UPLC liquid phase system nanoElute2 and then coupled with a mass spectrometer equipped with a nano-ion source (timsTOF Pro2) for data acquisition. Chromatographic separation was performed using a 75 μm ID × 15 cm reversed-phase chromatographic column. The mobile phase used was an acetonitrile-water-formic acid system, where mobile phase A was 0.1% formic acid aqueous solution and phase B was 0.1% formic acid acetonitrile solution. After the chromatographic column was equilibrated with 100% of phase A, the sample was directly loaded onto the chromatographic column by an autosampler and then gradient separated by the chromatographic column with a gradient duration of 45 min. The mass spectrometer used the DDAPaSEF mode for DDA data acquisition, and the scanning range was from 100 - 1700 m / z. During the PASEF MS / MS scanning process, the collision energy increased linearly with the ion mobility, from 20 eV (1 / K0 = 0.6 Vs / cm 2 ) to 59 eV (1 / K0 = 1.6 Vs / cm 2 ).

[0067] (4) As Figure 6 shown, by mass spectrometry analysis of the differential peptide segments between the coenzyme A treatment group and the control group, it was found that under the screening condition of P < 0.05, IGFBP2 was the protein with the highest differential multiple, indicating that coenzyme A treatment significantly increased the stability of IGFBP2 under proteases, suggesting that coenzyme A has a strong binding to IGFBP2.

[0068] Example 7: Knocking out IGFBP2 can improve the cell viability of cystine-deficient cardiomyocytes (1) Isolation and culture of primary cardiomyocytes from neonatal mice: The hearts of 1-day-old neonatal mice were cut with sterilized ophthalmic scissors, washed once with PBS, then minced, and digested with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37°C for 10 minutes each time, for a total of 5 times. After each digestion, the supernatant was transferred to a high-glucose DMEM solution containing 20% FBS for 1:1 neutralization. After digestion, the resulting solution containing cardiomyocytes was filtered through a 70 μm filter, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in a DMEM solution containing 10% FBS, and then the cells were transferred to a 10 cm cell culture dish and differentially adhered in a 37°C cell culture incubator for 90 min. The supernatant was collected, cell counting was performed, diluted with a DMEM solution containing 10% FBS, and 0.05 mM concentration of Brdu was added to inhibit the proliferation of fibroblasts. Subsequently, the cardiomyocytes were evenly seeded into 12-well plates at an inoculation density of 5×10 5 cells / well.

[0069] (2)IGFBP2 siRNA transfection: Take 3 μL of Lipofectamine™ (Thermo, 13778150) transfection reagent per well and place it in serum-free DMEM and mix well; take an appropriate amount of serum-free DMEM to dilute the IGFBP2 or control siRNA solution (20 nM), mix the diluted Lipofectamine™ and siRNA solution in a 1:1 ratio, gently mix and let stand at room temperature for 10 - 15 minutes to form a transfection complex. Remove the medium from the 12-well plate and wash the cells 2 times with PBS. Add the transfection complex to each well (adjust the final volume to 1 mL), and the final concentration of siRNA is 50 nM. Gently shake the 12-well plate to evenly distribute the transfection complex.

[0070] (3)Cystine deprivation-induced ferroptosis and coenzyme A intervention: 48 hours after siRNA transfection, replace the original medium with complete medium containing 10% FBS (normal control group) or cystine-free medium (model group), and cystine-free medium supplemented with coenzyme A (coenzyme A treatment group), and culture for 48 hours.

[0071] (4)Cell viability identification: Use the CCK8 kit (Beyotime, C0041) to evaluate the survival of neonatal rat cardiomyocytes.

[0072] (5)As Figure 7 shown, under the culture condition of cystine (-) + DMSO, the viability of primary neonatal rat cardiomyocytes in the control siRNA transfection group (siNC) was 25.80 ± 1.72%, which was significantly reduced compared with normal culture [Cystine (+)]. While in the IGFBP2 knockdown group (siIGFBP2), the survival rate of cardiomyocytes could be increased to 44.72 ± 4.56%, inhibiting the occurrence of ferroptosis in cardiomyocytes. There was no significant difference between the two groups under the treatment of coenzyme A, indicating that knocking down IGFBP2 could improve the viability of cystine-deprived cardiomyocytes, suggesting that coenzyme A might play an anti-death role by binding to the function of IGFBP2.

[0073] Example 8: Knocking out IGFBP2 can inhibit ferroptosis of cardiomyocytes caused by cystine deficiency (1)Isolation and culture of primary neonatal mouse cardiomyocytes: The hearts of 1-day-old neonatal mice were excised using sterile ophthalmic scissors. After washing once with PBS, the hearts were minced and digested with 0.05% trypsin + 0.05% type II collagenase at 37°C on a shaker for 10 minutes each time, for a total of 5 times. After each digestion, the supernatant was transferred to high-glucose DMEM solution containing 20% FBS for 1:1 neutralization. After digestion, the solution containing cardiomyocytes was filtered through a 70μm filter, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in DMEM solution containing 10% FBS, and then transferred to a 10 cm cell culture dish. The cells were differentially adhered in a 37°C cell culture incubator for 90 minutes. The supernatant was collected, cell counting was performed, and the cells were diluted with DMEM solution containing 10% FBS. Brdu at a concentration of 0.05 mM was added to inhibit the proliferation of fibroblasts. Subsequently, the cardiomyocytes were evenly seeded into a 12-well plate at an inoculation density of 5×10 5 cells / well.

[0074] (2)IGFBP2 siRNA transfection: Take 3 μL of Lipofectamine™ (Thermo, 13778150) transfection reagent per well and place it in serum-free DMEM for mixing; separately, dilute the IGFBP2 or control siRNA solution (20 nM) with an appropriate amount of serum-free DMEM. Mix the diluted Lipofectamine™ and siRNA solution in a 1:1 ratio, gently mix, and let it stand at room temperature for 15 minutes to form a transfection complex. Remove the medium from the 12-well plate and wash the cells 2 times with PBS. Add the transfection complex to each well (final volume adjusted to 1 mL), with a final siRNA concentration of 50 nM, and gently shake the 12-well plate to evenly distribute the transfection complex.

[0075] (3)Cystine deprivation-induced ferroptosis and coenzyme A intervention: 48 hours after siRNA transfection, the original medium was replaced with complete medium containing 10% FBS (normal control group) or cystine-free medium (model group), cystine-free medium supplemented with coenzyme A (coenzyme A treatment group), and cultured for 48 hours.

[0076] (4)Detection of 4HNE protein expression: Discard the culture medium, wash twice with pre-cooled PBS, add RIPA lysis buffer containing phosphatase inhibitor and protease inhibitor, and lyse on ice for 30 minutes. Quantify the protein using BCA protein assay kit. Add 5×loading buffer and heat the protein at 98°C in a metal bath for 30 minutes to denature it. Subsequently, perform immunoblotting experiments. The primary antibody is Anti-4 Hydroxynonenal antibody [Abcam, ab46545 (1:1000)], and the secondary antibody is Goat anti-rabbit IgG H&L [Abcam, ab205718 (1:10000)]. Detect the expression level of 4HNE protein to evaluate the effect of IGFBP2 knockdown on lipid peroxidation.

[0077] (5)As Figure 8 shown, after 48 hours of cystine deprivation, the expression of 4HNE in cardiomyocytes of the IGFBP2 knockdown model group [Cystine (-)] was significantly lower than that in the non-knockdown group [Cystine (-)], indicating that IGFBP2 knockout can inhibit the increase in intracellular lipid peroxide content after cystine deprivation. There was no significant change in the expression of 4HNE in cardiomyocytes with IGFBP2 knockdown in the coenzyme A treatment group [Cystine (-)+CoA], suggesting that IGFBP2 has a function of promoting ferroptosis, and coenzyme A exerts an anti-ferroptosis function in cardiomyocytes by binding to IGFBP2.

[0078] Unless otherwise specified, the raw materials and equipment used in the present invention are common raw materials and equipment in the art; the methods used in the present invention are conventional methods in the art unless otherwise specified.

[0079] The above are only the preferred embodiments of the present invention, and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent transformations made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of coenzyme A and / or agents that can promote intracellular coenzyme A synthesis and / or IGFBP2 inhibitors in the preparation of cardiomyocyte ferroptosis inhibitors.

2. Use of coenzyme A and / or agents that can promote intracellular coenzyme A synthesis and / or IGFBP2 inhibitors in the preparation of tool drugs that have the function of inhibiting myocardial cell ferroptosis.

3. Use of coenzyme A and / or agents that can promote intracellular coenzyme A synthesis and / or IGFBP2 inhibitors in the preparation of therapeutic drugs for myocardial cell ferroptosis-related diseases.

4. The use according to claim 3, characterized in that: The myocardial cell ferroptosis-related diseases include drug-induced heart failure, myocardial ischemia-reperfusion injury, septic cardiomyopathy, and diabetic cardiomyopathy.

5. The use according to claim 3, characterized in that: The therapeutic drug comprises at least one of coenzyme A, an agent capable of promoting the synthesis of coenzyme A in cells and an IGFBP2 inhibitor, and a pharmaceutically acceptable carrier, excipient or solvent.

6. Use of a drug containing coenzyme A and / or an agent that can promote intracellular coenzyme A synthesis and / or an IGFBP2 inhibitor for inhibiting in vitro myocardial cell ferroptosis, characterized in that: The coenzyme A inhibits in vitro cardiomyocyte ferroptosis by binding to IGFBP2.

7. A method for inhibiting myocardial cell ferroptosis in vitro, characterized in that: Drugs containing coenzyme A and / or reagents that can promote intracellular coenzyme A synthesis and / or IGFBP2 inhibitors are added to the in vitro cardiomyocyte culture system. Coenzyme A inhibits in vitro cardiomyocyte ferroptosis by binding to IGFBP2; IGFBP2 inhibitors inhibit in vitro cardiomyocyte ferroptosis by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

8. The use according to any one of claims 1 to 6 or the method according to claim 7, characterized in that: The reagent that can promote intracellular coenzyme A synthesis is a virus vector targeting myocardial cell adenocarcinoma inserted with PANK1 gene sequence; The IGFBP2 inhibitor is a small interfering RNA, an antisense oligonucleotide or an IGFBP2 neutralizing antibody directed against IGFBP2.

9. A method for constructing an in vitro cardiomyocyte model with low lipid peroxide accumulation, characterized in that: A cystine-deprived in vitro cardiomyocyte culture system is constructed, and drugs containing coenzyme A and / or reagents that can promote intracellular coenzyme A synthesis and / or IGFBP2 inhibitors are added at the same time. Coenzyme A reduces the accumulation of lipid peroxides in cardiomyocytes by binding to IGFBP2, thereby obtaining an in vitro cardiomyocyte model with low lipid peroxide accumulation; IGFBP2 inhibitors reduce the accumulation of lipid peroxides in cardiomyocytes by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2, thereby obtaining an in vitro cardiomyocyte model with low lipid peroxide accumulation.

10. A method for constructing an in vitro cardiomyocyte model with low lipid peroxide accumulation, characterized in that: The IGFBP2 gene of in vitro cardiomyocytes is first knocked out or knocked down, and then the in vitro cardiomyocytes are cultured in a cystine-free culture system, thereby obtaining an in vitro cardiomyocyte model with low lipid peroxide accumulation.

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