Use of coenzyme a in preparation of inhibitor of ferroptosis of myocardial cells

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to application of coenzyme A in preparation of a myocardial cell ferroptosis inhibitor. BACKGROUND

[0002] Ferroptosis is a new type of programmed cell death mode different from apoptosis, necrosis, pyroptosis and autophagy, which is closely related to cell metabolic disorder and redox imbalance. The main marker is iron-dependent lipid peroxide deposition in cells, and the cell morphology shows that the mitochondrial volume is reduced, the membrane density is increased, and the ridge is reduced.

[0003] At present, a large number of studies have shown that myocardial cell ferroptosis 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 the mouse models of ischemia-reperfusion injury and adriamycin-induced cardiomyopathy, myocardial cell ferroptosis characteristics are observed in heart tissue. The use of ferroptosis inhibitors can significantly improve the pathological damage of mouse heart and improve the survival rate of mice. Therefore, targeting myocardial cell ferroptosis is a potential effective means for treating heart disease.

[0004] Coenzyme A is a coenzyme containing adenosine diphosphate and pantothenic acid (vitamin B5) derivatives, which plays a key role in various metabolic reactions in the body. PANK1 is the first key enzyme for synthesizing coenzyme A. The main function of coenzyme A is to bind enzymes to promote the synthesis and decomposition of fatty acids, especially to participate in the process of beta-oxidation of fatty acids, which is one of the important ways of energy supply of the body. 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), and chronic liver. However, there is no research report on the effect of coenzyme A on myocardial cell ferroptosis, and the downstream molecular mechanism is not clear.

[0005] Insulin-like growth factor binding protein 2 (IGFBP2) is one of the members of the IGFBP family. The proteins in this family mainly participate in the regulation of 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 no research report on the effect of IGFBP2 on myocardial cell ferroptosis. SUMMARY

[0006] The application provides application of coenzyme A in preparation of a myocardial cell ferroptosis inhibitor. The application discloses for the first time that coenzyme A has the function of inhibiting myocardial cell ferroptosis, and finds that the function is achieved by binding IGFBP2 protein, so as to develop a new drug for targeted inhibition of myocardial cell ferroptosis, and provide a new theoretical basis and potential intervention strategy for myocardial injury repair.

[0007] The specific technical scheme of the application is:

[0008] In a first aspect, the application provides application of coenzyme A and / or an agent capable of promoting synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in preparation of a myocardial cell ferroptosis inhibitor.

[0009] The application finds for the first time that coenzyme A has the function of inhibiting myocardial cell ferroptosis. Specifically, the application finds through experiments that coenzyme A can bind IGFBP2, reduce accumulation of lipid peroxides in myocardial cells, and thus inhibit myocardial cell ferroptosis. Therefore, coenzyme A can be used to treat myocardial cell ferroptosis related diseases.

[0010] Similarly, the agent capable of promoting synthesis of coenzyme A in cells can overexpress genes related to synthesis of coenzyme A, so as to promote synthesis of coenzyme A in cells, increase the content of coenzyme A, and thus achieve an effect similar to that of exogenous coenzyme A. The IGFBP2 inhibitor can inhibit expression of IGFBP2 or inhibit function of IGFBP2, so as to achieve an effect similar to that of exogenous coenzyme A.

[0011] In a second aspect, the application provides application of coenzyme A and / or an agent capable of promoting synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in preparation of a tool drug having the function of inhibiting myocardial cell ferroptosis. The tool drug can be used for scientific research, for example, coenzyme A and / or the agent capable of promoting synthesis of coenzyme A in cells and / or the IGFBP2 inhibitor can be used to construct a related in vitro cell model in an in vitro experiment, based on the function of inhibiting myocardial cell ferroptosis.

[0012] In a third aspect, the application provides application of coenzyme A and / or an agent capable of promoting synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in preparation of a drug for treating myocardial cell ferroptosis related diseases.

[0013] Further, the myocardial cell ferroptosis related diseases include drug-induced heart failure, myocardial ischemia-reperfusion injury, septic cardiomyopathy, diabetic cardiomyopathy, etc.

[0014] As a preference, the drug includes at least one of coenzyme A, the agent capable of promoting synthesis of coenzyme A in cells and the IGFBP2 inhibitor, and a pharmaceutically acceptable carrier, excipient or solvent.

[0015] Further preferably, the therapeutic drug is an oral preparation or an injection preparation.

[0016] In a fourth aspect, the present application provides a use of a drug containing coenzyme A and / or an agent capable of promoting intracellular synthesis of coenzyme A and / or an IGFBP2 inhibitor in inhibiting iron death of myocardial cells in vitro: coenzyme A inhibits iron death of myocardial cells in vitro by binding to IGFBP2, and the IGFBP2 inhibitor inhibits iron death of myocardial cells in vitro by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

[0017] In a fifth aspect, the present application provides a method for inhibiting iron death of myocardial cells in vitro: adding a drug containing coenzyme A and / or an agent capable of promoting intracellular synthesis of coenzyme A and / or an IGFBP2 inhibitor to a culture system of myocardial cells in vitro, wherein coenzyme A inhibits iron death of myocardial cells in vitro by binding to IGFBP2. Specifically, the coenzyme A inhibits iron death of myocardial cells by reducing lipid peroxide accumulation in myocardial cells induced by cystine deprivation by binding to IGFBP2; and the IGFBP2 inhibitor inhibits iron death of myocardial cells in vitro by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

[0018] Preferably, the agent capable of promoting intracellular synthesis of coenzyme A is a targeted myocardial cell adenovirus vector inserted into the PANK1 gene sequence; and more preferably, the agent is PZ-2891.

[0019] Preferably, the IGFBP2 inhibitor is an IGFBP2 neutralizing antibody, a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO) against IGFBP2. The IGFBP2 neutralizing antibody can block the interaction of IGFBP2 with insulin-like growth factor (IGF) or other ligands by binding to IGFBP2, thereby inhibiting the function of IGFBP2. The small interfering RNA (siRNA) or the antisense oligonucleotide (ASO) against IGFBP2 can bind to IGFBP2 mRNA, thereby inhibiting the expression of IGFBP2.

[0020] Preferably, the drug comprises at least one of coenzyme A, an agent capable of promoting intracellular synthesis of coenzyme A, and an IGFBP2 inhibitor, and a pharmaceutically acceptable carrier, excipient or solvent.

[0021] Preferably, the effective concentration of coenzyme A in the culture system of myocardial cells in vitro is 50-500 μM.

[0022] In a sixth aspect, the present application provides a method for constructing an in-vitro model of myocardial cells with low lipid peroxide accumulation: an in-vitro culture system of cystine-deprived myocardial cells is constructed, and a drug containing coenzyme A and / or an agent that can promote the synthesis of intracellular coenzyme A and / or an IGFBP2 inhibitor (when the IGFBP2 inhibitor is small interfering RNA or antisense oligonucleotide against IGFBP2, the drug needs to be transfected in advance) is added, coenzyme A reduces lipid peroxide accumulation in myocardial cells by binding to IGFBP2, thereby obtaining an in-vitro model of myocardial cells with low lipid peroxide accumulation; the IGFBP2 inhibitor reduces lipid peroxide accumulation in myocardial cells by inhibiting the expression or function of IGFBP2, thereby obtaining an in-vitro model of myocardial cells with low lipid peroxide accumulation.

[0023] In a seventh aspect, the present application provides a method for constructing an in-vitro model of myocardial cells with low lipid peroxide accumulation: the IGFBP2 gene of in-vitro myocardial cells is first knocked out or knocked down, and then the in-vitro myocardial cells are cultured in a cystine-free culture system, thereby obtaining an in-vitro model of myocardial cells with low lipid peroxide accumulation.

[0024] Compared with the prior art, the present application has the beneficial effects that: the present application first proposes that coenzyme A can reduce ferroptosis of myocardial cells by binding to IGFBP2, provides the application of coenzyme A in the preparation of a myocardial cell ferroptosis inhibitor, and further provides the application of the preparation of a drug for treating myocardial cell ferroptosis-related diseases. It is expected to develop new drugs for targeting myocardial ferroptosis, and provide new theoretical basis and potential intervention strategies for myocardial injury repair. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The primary mouse myocardial cells were cultured in complete medium for 24 hours, and then replaced with complete medium [Cystine (+)], 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 [Cystine (-)+Fer-1]. After 48 hours, the cell morphology was observed under a light microscope, and the scale = 100 μm.

[0026] Figure 2Primary neonatal mouse cardiomyocytes were cultured in complete medium for 24 hours, then replaced with complete medium [Cystine (+)], cystine-free medium [Cystine (-)], cystine-free medium with 500 μΜ coenzyme A [Cystine (-) + CoA], and cystine-free medium with 1 μΜ Ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) as a positive control. Cell viability was detected after 48 hours, and the survival of cardiomyocytes was evaluated. The statistical results of the survival of cardiomyocytes in the four groups are shown. **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0027] Figure 3 Primary neonatal mouse cardiomyocytes were cultured in complete medium for 24 hours, then replaced with complete medium [Cystine (+)], cystine-free medium [Cystine (-)], cystine-free medium with 500 μΜ coenzyme A [Cystine (-) + CoA], and cystine-free medium with 1 μΜ Ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) as a positive control. Cell protein was collected after 36 hours, and Western Blot was used to detect the expression of 4HNE to evaluate the effect of coenzyme A on lipid peroxides. The statistical results of 4HNE expression in the three groups of cardiomyocytes (n = 6) are shown. *P < 0.05, ***P < 0.001.

[0028] Figure 4 Primary neonatal mouse cardiomyocytes were cultured in complete medium for 24 hours, then replaced with complete medium [Cystine (+)], cystine-free medium [Cystine (-)], cystine-free medium with 500 μΜ coenzyme A [Cystine (-) + CoA], and cystine-free medium with 1 μΜ Ferrostatin-1 (Fer-1, an inhibitor of ferroptosis) as a positive control. BODIPY 581 / 591 C11 (Invitrogen, D3861) staining was used after 48 hours, and flow cytometry was used to evaluate the effect of coenzyme A on lipid peroxides. ****P < 0.0001.

[0029] Figure 5 C57BL / 6 mice were injected with a myocardial cell-specific promoter PANK1 overexpression adeno-associated virus via the tail vein, and 4 weeks later, the mice were subjected to ischemia-reperfusion injury modeling. Small animal echocardiography was used to evaluate the effect of PANK1 overexpression on heart function in mice with ischemia-reperfusion injury before modeling (baseline) and at 1 day, 3 days, 7 days, and 28 days after modeling. *P < 0.05, **P < 0.01.

[0030] Figure 6 To screen the target proteins that can bind to coenzyme A in cardiomyocytes using Lip-MS technology.

[0031] Figure 7 Primary mouse cardiomyocytes were transfected with IGFBP2 siRNA or control reagent for 48 hours, and then replaced with complete culture medium [Cystine (+)] or cystine-free culture medium [Cystine (-)], cystine-free culture medium containing 500 μM coenzyme A [Cystine (-) + CoA]. After 48 hours, cell viability was detected to evaluate the survival of cardiomyocytes. **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Figure 8 Primary mouse cardiomyocytes were transfected with IGFBP2 siRNA or control reagent for 48 hours, and then replaced with complete culture medium [Cystine (+)] or cystine-free culture medium [Cystine (-)], cystine-free culture medium containing 500 μM coenzyme A [Cystine (-) + CoA]. After 48 hours, cell proteins were collected, and Western Blot was used to detect the expression of IGFBP2 and 4HNE to evaluate the effect of IGFBP2 knockout on lipid peroxidation in cardiomyocytes. DETAILED DESCRIPTION

[0033] The application will be further described below in conjunction with examples.

[0034] OVERALL EXAMPLE

[0035] In a first aspect, the application provides use of coenzyme A and / or an agent that can promote synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in the preparation of an inhibitor of ferroptosis in cardiomyocytes.

[0036] In a second aspect, the application provides use of coenzyme A and / or an agent that can promote 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.

[0037] In a third aspect, the application provides use of coenzyme A and / or an agent that can promote synthesis of coenzyme A in cells and / or an IGFBP2 inhibitor in the preparation of a therapeutic drug for a ferroptosis-related disease in cardiomyocytes.

[0038] Further, the ferroptosis-related disease in cardiomyocytes includes drug-induced heart failure, myocardial ischemia-reperfusion injury, septic cardiomyopathy, diabetic cardiomyopathy, etc.

[0039] In some preferred embodiments, the therapeutic drug comprises at least one of coenzyme A, an agent that can promote the synthesis of intracellular coenzyme A, and an IGFBP2 inhibitor, and a pharmaceutically acceptable carrier, excipient or solvent.

[0040] In some more preferred embodiments, the therapeutic drug is an oral preparation or an injection preparation.

[0041] In a fourth aspect, the present application provides a use of a drug containing coenzyme A or an agent that can promote the synthesis of intracellular coenzyme A and / or an IGFBP2 inhibitor in inhibiting the death of myocardial cells in vitro: the coenzyme A inhibits the death of myocardial cells in vitro by binding to IGFBP2; and the IGFBP2 inhibitor inhibits the death of myocardial cells in vitro by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

[0042] In a fifth aspect, the present application provides a method for inhibiting the death of myocardial cells in vitro: adding a drug containing coenzyme A and / or an agent that can promote the synthesis of intracellular coenzyme A and / or an IGFBP2 inhibitor to a culture system of myocardial cells in vitro, the coenzyme A inhibits the death of myocardial cells in vitro by binding to IGFBP2. Specifically, the coenzyme A inhibits the death of myocardial cells by reducing the accumulation of lipid peroxides in myocardial cells induced by cystine deprivation through binding to IGFBP2; and the IGFBP2 inhibitor inhibits the death of myocardial cells in vitro by inhibiting the expression of IGFBP2 or inhibiting the function of IGFBP2.

[0043] In some preferred embodiments, the agent that can promote the synthesis of intracellular coenzyme A is a targeted myocardial cell adenovirus vector inserted into the PANK1 gene sequence; and more preferably, PZ-2891.

[0044] In some preferred embodiments, the IGFBP2 inhibitor is an IGFBP2 neutralizing antibody (further preferably, Qiagen, item number QC-5842R), a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO) against IGFBP2. The IGFBP2 neutralizing antibody can block the interaction of IGFBP2 with insulin-like growth factor (IGF) or other ligands by binding to IGFBP2, thereby inhibiting the function of IGFBP2. The small interfering RNA (siRNA) or the antisense oligonucleotide (ASO) against IGFBP2 can bind to IGFBP2 mRNA, thereby inhibiting the expression of IGFBP2. In some preferred embodiments, the drug comprises at least one of coenzyme A, an agent that can promote the synthesis of intracellular coenzyme A, and an IGFBP2 inhibitor, and a pharmaceutically acceptable carrier, excipient or solvent.

[0045] In some preferred embodiments, the effective concentration of coenzyme A in the in vitro myocardial cell culture system is 50-500 μM.

[0046] In a sixth aspect, the present application provides a method for constructing an in vitro model of myocardial cells with low lipid peroxide accumulation: an in vitro culture system of cystine-deprived myocardial cells is constructed, and a drug containing coenzyme A and / or an agent that can promote the synthesis of intracellular coenzyme A and / or an IGFBP2 inhibitor (when the IGFBP2 inhibitor is small interfering RNA or antisense oligonucleotide against IGFBP2, the drug needs to be transfected in advance) is added. Coenzyme A reduces lipid peroxide accumulation in myocardial cells by binding to IGFBP2, thereby obtaining an in vitro model of myocardial cells with low lipid peroxide accumulation. The IGFBP2 inhibitor reduces lipid peroxide accumulation in myocardial cells by inhibiting the expression or function of IGFBP2, thereby obtaining an in vitro model of myocardial cells with low lipid peroxide accumulation.

[0047] In a seventh aspect, the present application provides a method for constructing an in vitro model of myocardial cells with low lipid peroxide accumulation: the IGFBP2 gene of in vitro myocardial cells is first knocked out or knocked down, and then the in vitro myocardial cells are cultured in a cystine-free culture system, thereby obtaining an in vitro model of myocardial cells with low lipid peroxide accumulation. Specific embodiments

[0049] Example 1: Coenzyme A treatment can inhibit morphological changes of myocardial cells caused by cystine deficiency

[0050] (1) Isolation and culture of primary myocardial cells of milk mice: The heart of a 1-day-old milk mouse was cut off with sterilized ophthalmic scissors, washed once with PBS, and then cut into pieces. The pieces were digested with 0.05% trypsin and 0.05% collagenase II at 37°C on a shaker for 10 minutes each time, a total of 5 times. After each digestion, the supernatant was transferred to a 1:1 neutralization solution containing 20% FBS in high-glucose DMEM. After the digestion was completed, the resulting solution containing myocardial cells was filtered with a 70-μm filter, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in a DMEM solution containing 10% FBS, and then transferred to a 10-cm cell culture dish. The cells were incubated in a 37°C cell incubator for 90 min, and the supernatant was collected. The cells were counted, diluted with a DMEM solution containing 10% FBS, and 0.05 mM Brdu was added to inhibit the proliferation of fibroblasts. Subsequently, the myocardial cells were uniformly seeded into a 12-well plate at a seeding density of 5 x 10 5

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

[0052] (3) Observe and record cell morphology under a light microscope.

[0053] (4) such as Figure 1 As shown, under cystine-free culture conditions [Cystine (-) + DMSO], cardiomyocytes in the normal control group [Cystine (+) + DMSO] showed significant shrinkage, reduced volume, and cell death after 48 hours. However, cardiomyocyte morphology significantly improved after coenzyme A treatment [Cystine (-) + CoA] compared to the cystine-free group [Cystine (-) + DMSO]. This indicates that coenzyme A treatment can inhibit the morphological changes in cardiomyocytes caused by cystine deficiency.

[0054] Example 2: Coenzyme A has a regulatory effect on cardiomyocyte survival.

[0055] (1) Isolation and culture of primary cardiomyocytes from newborn mice: The hearts of newborn mice within 1 day of birth were cut off with sterile ophthalmic scissors, washed with PBS, 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 70μm filter, centrifuged at 1000rpm for 5min, and the supernatant was discarded. The cells were resuspended in a high-glucose DMEM solution containing 10% FBS, and then transferred to 10cm cell culture dishes. The cells were cultured at 37°C for 90min under differential adhesion. The supernatant was collected, and the cells were counted. The cells were diluted with a high-glucose DMEM solution containing 10% FBS, and 0.05mM BrdU was added to inhibit fibroblast proliferation. The cardiomyocytes were then evenly inoculated into 96-well plates at a seeding density of 5×10⁶ cells / well. 4 Cells / well

[0056] (2) Cystine deprivation-induced ferroptosis and coenzyme A intervention: The culture medium of primary cardiomyocytes was replaced with complete culture medium containing 10% FBS (normal control group) or cystine-free culture medium (model group), cystine-free culture medium with added coenzyme A (coenzyme A treatment group), or cystine-free culture medium with added Fer-1 (positive treatment group), and cultured for 48 hours.

[0057] (3) Cell viability assessment: The viability of neonatal rat cardiomyocytes was evaluated using the CCK8 kit (Beyotime, C0041).

[0058] (4) such as Figure 2 As shown, under cystine-free culture conditions [Cystine (-) + DMSO], the viability of primary cardiomyocytes from the model group of suckling rats was 7.03 ± 1.43%, which was significantly lower than that under normal culture conditions [Cystine (+)]. However, the addition of Fer-1 [Cystine (-) + Fer-1] increased the cardiomyocyte survival rate to 67.02 ± 10.98% and inhibited ferroptosis in cardiomyocytes. Coenzyme A treatment [Cystine (-) + CoA] increased the cardiomyocyte survival rate to 63.15 ± 11.40%, indicating that coenzyme A treatment can inhibit cystine deprivation-induced cardiomyocyte death, suggesting that coenzyme A has a direct regulatory effect on cardiomyocyte survival.

[0059] Example 3: Coenzyme A treatment can reduce the expression of 4HNE protein in cardiomyocytes.

[0060] (1) Isolation and culture of primary cardiomyocytes from newborn mice: The hearts of newborn mice were cut off with sterile ophthalmic scissors on the first day of life, washed with PBS, 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 DMEM solution containing 20% ​​FBS at a 1:1 ratio for neutralization. After digestion, the resulting solution containing cardiomyocytes was filtered through a 70μm filter, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended in DMEM solution containing 10% FBS. The cells were then transferred to 10cm cell culture dishes and cultured at 37°C for 90min under differential adhesion. The culture dishes were then removed, the supernatant was collected, and the cells were counted. The cells were diluted with DMEM solution containing 10% FBS, and 0.05mM BrdU was added to inhibit fibroblast proliferation. The cells were then evenly inoculated into 12-well plates at a seeding density of 5×10⁶ cells / well. 5 Cells / well

[0061] (2) Cystine deprivation-induced ferroptosis and coenzyme A intervention: The culture medium of primary cardiomyocytes was replaced with complete culture medium containing 10% FBS (normal control group) or cystine-free culture medium (model group), cystine-free culture medium with added coenzyme A (coenzyme A treatment group), or cystine-free culture medium with added Fer-1 (positive treatment group), and cultured for 48 hours.

[0062] (3) 4HNE protein expression detection: discard the culture medium, wash twice with pre-cooled PBS, add RIPA lysis buffer containing phosphatase inhibitor and protease inhibitor, lyse on ice for 30 minutes, BCA protein quantification, add 5x loading, heat denatured protein at 98°C metal bath for 30 minutes. Then perform immunoblotting experiment, primary antibody Anti-4 Hydroxynonenal antibody (Abeam, ab46545, 1:1000), secondary antibody Goat Anti-Rabbit IgG H&L (Abeam, ab205718, 1:10000), detect the expression content of 4HNE protein, evaluate the effect of coenzyme A on lipid peroxide.

[0063] (4) As shown in Figure 3 Figure 4, after 48 hours of cystine deprivation, the 4HNE expression of the myocardial cells in the modeling group [Cystine (-)] was significantly higher than that in the normal control group [Cystine (+)], indicating that the lipid peroxide content in the cells after cystine deprivation was significantly increased, and the 4HNE expression of the coenzyme A treatment group [Cystine (-) + CoA] was significantly lower than that of the modeling group, indicating that coenzyme A treatment can significantly reduce the accumulation of lipid peroxide after cystine deprivation, and coenzyme A treatment can inhibit myocardial cell ferroptosis.

[0064] Example 4: Coenzyme A treatment can inhibit myocardial cell lipid peroxidation

[0065] (1) Isolation and culture of primary myocardial cells of milk mice: Sterile ophthalmic scissors were used to cut the heart of 1-day-old milk mice, washed once with PBS, cut into small pieces, and digested with 0.05% trypsin + 0.05% collagenase II at 37°C on a shaker for 10 minutes each time, a total of 5 times. After each digestion, the supernatant was transferred to a 1:1 neutralization solution containing 20% FBS in high glucose DMEM. After digestion was completed, the resulting solution containing myocardial cells was filtered with a 70μm filter, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were resuspended with DMEM containing 10% FBS. The cells were then transferred to a 10cm cell culture dish, and the cells were incubated at 37°C in a cell incubator for 90min. The supernatant was collected, and the cells were counted and diluted with 10% FBS in DMEM. Brdu was added at a concentration of 0.05mM to inhibit the proliferation of fibroblasts. The myocardial cells were then evenly seeded into a 6-well plate at a seeding density of 1x10 6

[0066] ​(2) Cystine deprivation-induced ferroptosis and coenzyme A intervention: The culture medium of primary cardiomyocytes was replaced with complete culture medium containing 10% FBS (normal control group) or cystine-free culture medium (model group), cystine-free culture medium with added coenzyme A (coenzyme A treatment group), or cystine-free culture medium with added Fer-1 (positive treatment group), and cultured for 48 hours.

[0067] (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 of each group by flow cytometry.

[0068] (4) such as Figure 4 As shown, after 48 hours of cystine deprivation, flow cytometry analysis revealed that the average fluorescence intensity of the FITC channel in the model group [Cystine (-)] was significantly higher than that in the normal control group [Cystine (+)], indicating a significant increase in intracellular lipid peroxide content after cystine deprivation. In contrast, the average fluorescence intensity of the FITC channel in the coenzyme A treatment group [Cystine (-)+CoA] was significantly lower than that in the model group, suggesting that coenzyme A treatment can significantly reduce the accumulation of lipid peroxides after cystine deprivation and inhibit ferroptosis in cardiomyocytes.

[0069] Example 5: Overexpression of coenzyme A synthase PANK1 can improve cardiac function in mice with myocardial ischemia-reperfusion injury.

[0070] (1) PANK1 overexpressing adeno-associated virus (vector name GV57; element sequence: cTNTp-MCS-3Flag-T2A-EGFP; sequence is the CDS sequence of the NM_001114339.2 gene) was injected via tail vein: 4-week-old male C57BL / 6 mice were selected, and each mouse was injected with 150 μL containing 3×10 11 Mice injected with a viral load of physiological saline and then used as the control group were set up as the control group.

[0071] (2) Mouse heart ischemia-reperfusion injury modeling: Four weeks after adeno-associated virus injection, mice were anesthetized with intraperitoneal injection of chloral hydrate (100 mg / kg). After anesthesia, tracheal intubation was performed and connected to a small animal respirator. The chest wall was incised at the 4th intercostal space on the left side, and the heart and left anterior descending branch of the left coronary artery (LAD) were exposed. A 6-0 silk thread was carefully threaded through the LAD and a slipknot was tied below it. When the slipknot was tied, the color of the left ventricular myocardium was pale, indicating successful ischemia, and the ischemia time was set to 45 minutes. Then the slipknot was loosened to restore coronary blood flow, and reperfusion was completed. After the operation, the chest wall and skin were sutured, and postoperative warming and nursing were given. The sham group (Sham) mice only had the silk thread threaded through the LAD and then removed immediately, without ligation.

[0072] (3) Mouse heart function evaluation: The mice were placed on the ultrasonic imaging platform, fixed with the back down, and the ultrasonic coupling agent without air bubbles was applied to the shaved area on the chest. The MS400 probe (center frequency 30 MHz) of the VEVO 3100 system (FUJIFILM Visual Sonics, Canada) was used to obtain long-axis and short-axis views. In the long-axis view of M-mode ultrasound, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and other conditions were recorded and analyzed. The results showed that overexpression of PANK1 to promote endogenous synthesis of coenzyme A in cardiomyocytes could improve the heart function of mice with ischemia-reperfusion injury

[0073] Example 6: Mass spectrometry screening found that IGFBP2 is a binding target of coenzyme A

[0074] (1) Isolation and culture of primary myocardial cells of milk mice: Sterile ophthalmic scissors were used to cut the heart of 1-day-old milk mice. After washing with PBS, the heart was cut into small pieces and digested with 0.05% trypsin and 0.05% collagenase II at 37°C on a shaker for 10 minutes each time, 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 was completed, the resulting solution containing myocardial cells was filtered with a 70-μm filter, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in a DMEM solution containing 10% FBS. After that, the cells were transferred to a 10-cm cell culture dish, and the cells were incubated in a 37°C cell incubator for 90 min. The supernatant was collected, and the cells were counted and diluted with a DMEM solution containing 10% FBS. Brdu was added at a concentration of 0.05 mM to inhibit the proliferation of fibroblasts. Subsequently, the myocardial cells were evenly inoculated into a 10-cm cell culture dish at a seeding density of 6 x 10 6 cells / dish.

[0075] (2) Cell sample preparation: After 24 hours of culture, the cells were washed twice with pre-cooled PBS, then scraped off with a spatula, centrifuged, and the supernatant discarded. The cell pellet was quickly frozen with liquid nitrogen. The sample was added to mass spectrometry water, ground in a low-temperature grinder, and ultrasonicated in an ice water bath for 20 min. Centrifuged at 12000 rpm, 4°C for 10 min, and the supernatant was transferred to a new EP tube. BCA quantification was performed. 600 μg of the extracted total protein was divided into 6 portions, with 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 coenzyme A 33 mMol as the experimental group (D1, D2, D3). PK was added at a ratio of 1:100, incubated at room temperature for 5 min, incubated at 95°C for 5 min, and then naturally cooled to room temperature. 2% SDC and 200 mM ABC were added. After trypsin degradation and peptide elution, the sample was detected on the instrument.

[0076] (3) nano-LC-MS / MS detection: 2 μL of total peptide was taken from each sample, separated by nano-UPLC liquid phase system anoElute2, and then detected by mass spectrometer (timsTOF Pro2) equipped with a nanometer ion source. Chromatographic separation was performed using a 75 μm ID x 15 cm reversed-phase chromatographic column. The mobile phase used acetonitrile-water-formic acid system, in which the mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was 0.1% formic acid acetonitrile solution. After the chromatographic column was equilibrated with 100% A phase, the sample was directly injected into the chromatographic column by the automatic sampler, and then separated by gradient elution on the chromatographic column. The gradient time was 45 min. The mass spectrometer was used for DDA data acquisition in DDAPaSEF mode, 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 ).

[0077] (4) As shown in Figure 6 , by analyzing the differential peptides of the coenzyme A treatment group and the control group by mass spectrometry, it was found that under the screening condition of P < 0.05, IGFBP2 was the protein with the highest differential fold, indicating that coenzyme A treatment significantly increased the stability of IGFBP2 under protease, indicating that coenzyme A had strong binding with IGFBP2.

[0078] Example 7: Knockout of IGFBP2 can improve the cell viability of cystine-deficient cardiomyocytes

[0079] (1) Neonatal mouse primary cardiomyocyte isolation and culture: The heart of 1-day-old neonatal mice was cut off with sterilized ophthalmic scissors, washed once with PBS, and then cut into small pieces. The pieces were digested with 0.05% trypsin and 0.05% collagenase II at 37°C on a shaker for 10 minutes each time, 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 the digestion was completed, the resulting solution containing cardiomyocytes was filtered with a 70-μm filter, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended with a DMEM solution containing 10% FBS. The cells were then transferred to a 10-cm cell culture dish, and the cells were allowed to adhere for 90 min in a 37°C cell incubator. The supernatant was collected, and the cells were counted. The cells were diluted with a DMEM solution containing 10% FBS, and Brdu was added at a concentration of 0.05 mM to inhibit the proliferation of fibroblasts. The cardiomyocytes were then evenly distributed into 12-well plates at a seeding density of 5 x 10 5

[0080] (2) IGFBP2 siRNA transfection: Lipofectamine™ (Thermo, 13778150) transfection reagent was taken in an amount of 3 μL per well, and was mixed in serum-free DMEM. An appropriate amount of serum-free DMEM was used to dilute IGFBP2 or control siRNA solution (20 nM). The diluted Lipofectamine™ and siRNA solution were mixed at a ratio of 1:1, gently mixed, and then incubated at room temperature for 10-15 minutes to form a transfection complex. The culture medium in the 12-well plate was removed, and the cells were washed twice with PBS. The transfection complex was added to each well (the final volume was adjusted to 1 mL), and the final concentration of siRNA was 50 nM. The 12-well plate was gently shaken to evenly distribute the transfection complex.

[0081] (3) Cysteine deprivation-induced ferroptosis and coenzyme A intervention: After 48 hours of siRNA transfection, the original culture medium was replaced with complete culture medium containing 10% FBS (normal control group) or cysteine-free culture medium (model group), or cysteine-free culture medium supplemented with coenzyme A (coenzyme A treatment group), and cultured for 48 hours.

[0082] (4) Cell viability identification: The CCK8 kit (Beyotime, C0041) was used to evaluate the survival of the neonatal rat cardiomyocytes.

[0083] (5) As Figure 7 ​As shown, the viability of the transfected control siRNA group (siNC) neonatal rat primary myocardial cells under cystine (-) + DMSO culture conditions was 25.80 ± 1.72%, which was significantly lower than that under normal culture conditions [cystine (+)], while the addition of the IGFBP2 knockdown group (siIGFBP2) increased the survival rate of myocardial cells to 44.72 ± 4.56%, inhibiting the occurrence of myocardial cell ferroptosis, and there was no significant difference between the two groups under coenzyme A treatment, indicating that knocking down IGFBP2 can improve the viability of cystine-deprived myocardial cells, suggesting that coenzyme A may exert an anti-death effect by binding to the function of IGFBP2.

[0084] Example 8: Knocking out IGFBP2 can inhibit myocardial cell ferroptosis caused by cystine deficiency

[0085] (1) Isolation and culture of neonatal mouse primary myocardial cells: The heart of a 1-day-old neonatal mouse was cut off with sterilized ophthalmic scissors, washed once with PBS, and then cut into small pieces. The pieces were digested with 0.05% trypsin and 0.05% collagenase II 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 a 1:1 mixture of high-glucose DMEM solution containing 20% FBS for neutralization. After the digestion was completed, the resulting solution containing myocardial cells was filtered through a 70-μm filter, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in a DMEM solution containing 10% FBS, and then transferred to a 10-cm cell culture dish. The cells were incubated in a 37°C cell incubator for 90 min, and then the supernatant was collected. The cells were diluted with a DMEM solution containing 10% FBS, and 0.05 mM Brdu was added to inhibit the proliferation of fibroblasts. The myocardial cells were then evenly distributed into 12-well plates at a seeding density of 5 × 10 5

[0086] (2) IGFBP2 siRNA transfection: 3 μL of Lipofectamine™ (Thermo, 13778150) transfection reagent was taken and mixed in serum-free DMEM; an appropriate amount of serum-free DMEM was used to dilute the IGFBP2 or control siRNA solution (20 nM), and the diluted Lipofectamine™ and siRNA solution were mixed at a ratio of 1:1, gently mixed, and then incubated at room temperature for 15 min to form a transfection complex. The culture medium in the 12-well plate was removed, and the cells were washed twice with PBS. The transfection complex was added to each well (the final volume was adjusted to 1 mL), and the final concentration of siRNA was 50 nM. The 12-well plate was gently shaken to evenly distribute the transfection complex.

[0087] ​(3) Cystine deprivation induced ferroptosis and coenzyme A intervention: 48 hours after siRNA transfection, the original culture medium was replaced with complete culture medium containing 10% FBS (normal control group) or cystine-free medium (modeling group), cystine-free medium added with coenzyme A (coenzyme A treatment group), and cultured for 48 hours.

[0088] (4) 4HNE protein expression detection: the culture medium was discarded, and the cells were washed twice with pre-cooled PBS, and then lysed with RIPA lysis buffer containing phosphatase inhibitor and protease inhibitor on ice for 30 minutes. BCA protein quantification was performed, 5x loading was added, and the protein was denatured at 98°C metal bath for 30 minutes. Subsequently, Western blotting was performed, the first antibody was Anti-4 Hydroxynonenal antibody [Abcam, ab46545 (1:1000)], the second antibody was Goat Anti-Rabbit IgG H&L [Abcam, ab205718 (1:10000)], and the expression content of 4HNE protein was detected to evaluate the effect of IGFBP2 knockdown on lipid peroxide.

[0089] (5) As shown in Figure 8 , after 48 hours of cystine deprivation, the 4HNE expression of the IGFBP2-knocked-down myocardial cells in the modeling group [Cystine (-)] was significantly lower than that in the non-knocked-down group [Cystine (-)], indicating that IGFBP2 knockdown can inhibit the increase in intracellular lipid peroxide content after cystine deprivation. The IGFBP2-knocked-down myocardial cells in the coenzyme A treatment group [Cystine (-)+CoA] showed no significant change in 4HNE expression, indicating that IGFBP2 has a ferroptosis-promoting function, and coenzyme A can play a role in resisting myocardial cell ferroptosis by binding to IGFBP2.

[0090] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified; the methods used in the present application are conventional methods in the art unless otherwise specified.

[0091] The above is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change and equivalent transformation of the above embodiment based on the technical essence of the present application are still within the protection scope of the technical solution of the present application.

Claims

1. Use of coenzyme A in the preparation of a drug for treating a myocardial cell ferroptosis-related disease, characterized in that: The myocardial cell ferroptosis related disease is drug-induced heart failure.

2. Use of coenzyme A in the preparation of a drug for treating a myocardial cell ferroptosis-related disease, characterized in that: The myocardial cell ferroptosis related disease is myocardial ischemia-reperfusion injury.

3. Use of coenzyme A in the preparation of a drug for treating a myocardial cell ferroptosis-related disease, characterized in that: The myocardial cell ferroptosis related disease is septic cardiomyopathy.

4. Use of coenzyme A in the preparation of a drug for treating a myocardial cell ferroptosis-related disease, characterized in that: The myocardial cell ferroptosis related disease is diabetic cardiomyopathy.

5. Use according to any one of claims 1 to 4, characterized in that: The therapeutic drug comprises coenzyme A, and a pharmaceutically acceptable carrier, excipient or solvent.

6. Use according to claim 5, characterized in that: The therapeutic drug is an oral preparation.

7. Use according to claim 5, characterized in that: The therapeutic drug is an injection preparation.

8. Use according to any one of claims 1 to 4, characterized in that: The coenzyme A has an effect of inhibiting myocardial cell ferroptosis.

9. Use according to any one of claims 1 to 4, characterized in that: The coenzyme A has an effect of inhibiting IGFBP2.

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