Application of inosine in preparation of myocardial cell ferroptosis inhibitor

Abstract

The invention relates to the field of biological medicine, and discloses application of inosine in preparation of a myocardial cell ferroptosis inhibitor. The invention discloses that inosine has the function of inhibiting myocardial ferroptosis for the first time, so that a new drug is expected to be developed for targeting myocardial 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 in particular to the application of inosine in the preparation of a myocardial cell ferroptosis inhibitor. Background Art

[0002] Ferroptosis is a new type of programmed cell death discovered in recent years, which is different from apoptosis, necrosis and autophagy. It is closely related to cell metabolism, redox and other processes. Its main sign is the deposition of iron-dependent lipid peroxides in cells, and its cell morphology is manifested by reduced mitochondrial volume, increased membrane density and reduced cristae.

[0003] At present, more and more studies have shown that cardiomyocyte ferroptosis is closely related to the occurrence and development of various heart diseases. For example, in the mouse models of ischemia-reperfusion injury and doxorubicin-induced cardiomyopathy, the heart tissue showed obvious characteristics of cardiomyocyte ferroptosis. Treatment with ferroptosis inhibitors can significantly improve the pathological damage of the mouse heart and increase the survival rate of mice. Therefore, inhibiting cardiomyocyte ferroptosis is currently a promising way to treat heart disease.

[0004] Inosine is a nucleoside compound formed by the combination of hypoxanthine and ribose, and has good cell membrane permeability. In the body, inosine is mainly involved in the synthesis of purine nucleotides and post-transcriptional regulation of RNA. At present, inosine is mainly used in the clinic to treat leukopenia, thrombocytopenia and other symptoms, and is also used as an adjuvant treatment for liver damage and heart disease. In the adjuvant treatment of heart disease, inosine mainly plays a role in dilating coronary arteries and exerting positive inotropic effects, thereby improving patient symptoms. However, there is currently no research to prove the direct effect of inosine on the survival of myocardial cells. Summary of the invention

[0005] The present invention provides the use of inosine in the preparation of a myocardial cell ferroptosis inhibitor. The present invention discloses for the first time that inosine has the function of inhibiting myocardial cell ferroptosis, and is therefore expected to develop new drugs targeting myocardial ferroptosis, providing a new theoretical basis and potential intervention strategy for myocardial injury repair.

[0006] The specific technical scheme of the present invention is: In a first aspect, the present invention provides the use of inosine in preparing an inhibitor of myocardial cell ferroptosis.

[0007] The present invention finds that at the cellular level, the concentration of inosine that exerts an anti-cardiomyocyte ferroptosis effect is 100 μM. The present invention discloses for the first time that inosine has the function of inhibiting cardiomyocyte ferroptosis. Specifically, the present invention finds through experiments that inosine can significantly reduce cardiomyocyte ferroptosis induced by cystine deprivation, and finds that inosine can significantly reduce the accumulation of lipid peroxides in cardiomyocytes induced by cystine deprivation. Therefore, inosine can be used to treat diseases related to cardiomyocyte ferroptosis.

[0008] In a second aspect, the present invention provides the use of inosine in preparing a drug for treating diseases related to myocardial cell ferroptosis.

[0009] In a third aspect, the present invention provides the use of an agent that can promote the synthesis of inosine in vivo in the preparation of an inhibitor of myocardial cell ferroptosis.

[0010] The reagent that can promote the synthesis of inosine in the body can overexpress the genes related to the synthesis of inosine, thereby promoting the synthesis of inosine in the body and increasing the inosine content in the body, thereby achieving an effect similar to that of exogenous inosine intake.

[0011] In a fourth aspect, the present invention provides the use of an agent that can promote the synthesis of inosine in vivo in the preparation of a drug for treating diseases related to myocardial cell ferroptosis.

[0012] Preferably, the agent capable of promoting the inosine synthesis in vivo is selected from an adeno-associated virus vector inserted with an adenosine deaminase gene or an extracellular 5'-nucleotidase gene.

[0013] Preferably, the therapeutic drug comprises inosine and / or an agent that can promote the synthesis of inosine in vivo and a pharmaceutically acceptable carrier, excipient or solvent.

[0014] Furthermore, the therapeutic drug is an injectable preparation.

[0015] Compared with the prior art, the beneficial effects of the present invention are: the present invention proposes for the first time that inosine can reduce myocardial cell ferroptosis, provides the application of inosine as and in the preparation of myocardial cell ferroptosis inhibitors, and provides the application of inosine in the preparation of drugs for treating myocardial cell ferroptosis-related diseases. It is expected to develop new drugs targeting myocardial ferroptosis and provide a new theoretical basis and potential intervention strategy for myocardial injury repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Primary neonatal mouse cardiomyocytes were treated with inosine or its control solvent DMSO for 24 hours, and then replaced with complete medium [Cystine (+)] or cystine-free medium [Cystine (-)]. Cell morphology was observed under a light microscope 48 hours later. Scale bar = 100 μm; Figure 2For primary neonatal mouse cardiomyocytes, they were treated with inosine or its control solvent for 24 hours, and then replaced with complete medium [Cystine (+)] or cystine-free medium [Cystine (-)], and cystine-free medium containing 1μM Ferrostatin-1 (Fer-1, ferroptosis inhibitor) as a positive control. After 48 hours, cell viability was detected to evaluate cardiomyocyte survival. Statistical results of cardiomyocyte survival in the four groups. ***P<0.001, ****P<0.0001; Figure 3 Primary neonatal mouse cardiomyocytes were treated with inosine or its control solvent for 24 hours, and then replaced with complete medium [Cystine (+)] or cystine-free medium [Cystine (-)]. Cell proteins were collected 36 hours later, and the expression of 4HNE was detected by Western Blot to evaluate the effect of inosine on lipid peroxides. Statistical results of 4HNE expression in three groups of cardiomyocytes (n=6), *P<0.05, ***P<0.001; Figure 4 Primary neonatal mouse cardiomyocytes were treated with inosine or its control solvent for 24 hours, and then replaced with complete medium [Cystine (+)] or cystine-free medium [Cystine (-)], as well as cystine-free medium containing 1 μM Ferrostatin-1 (Fer-1, ferroptosis inhibitor) as a positive control. After 48 hours, BODIPY 581 / 591C11 (Invitrogen, D3861) staining was used to evaluate the effect of inosine on lipid peroxidation by flow cytometry. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with the embodiments.

[0018] Overall embodiment The present invention provides the use of inosine or a reagent capable of promoting the synthesis of inosine in vivo in the preparation of an inhibitor of myocardial cell ferroptosis.

[0019] The present invention provides the use of inosine or a reagent capable of promoting the synthesis of inosine in vivo in the preparation of a drug for treating diseases related to myocardial cell ferroptosis.

[0020] Preferably, the agent capable of promoting the inosine synthesis in vivo is selected from an adeno-associated virus vector inserted with an adenosine deaminase gene or an extracellular 5'-nucleotidase gene.

[0021] Preferably, the therapeutic drug comprises inosine and / or an agent that can promote the synthesis of inosine in vivo and a pharmaceutically acceptable carrier, excipient or solvent. Further, the therapeutic drug comprising inosine is an oral preparation or an injection preparation, and the therapeutic drug comprising an agent that can promote the synthesis of inosine in vivo is an injection preparation.

[0022] Example 1 (1) Isolation and culture of primary cardiomyocytes from suckling mice: The hearts of one-day-old suckling mice were cut with sterile ophthalmic scissors, washed with PBS, chopped, and digested with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37°C for 10 min each time, for a total of 5 times. After each digestion, the supernatant was transferred to a high-glucose DMEM solution containing 20% ​​FBS and neutralized in a 1:1 ratio. After digestion, the resulting solution containing cardiomyocytes was filtered with a 70 μm filter and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended in a DMEM solution containing 10% FBS. The cells were then transferred to a 10 cm cell culture dish and allowed to adhere differentially in a cell culture incubator at 37°C for 90 min. The supernatant was collected, the cells were counted, and diluted with a DMEM solution containing 10% FBS. 0.05 mM Brdu was added to inhibit the proliferation of fibroblasts. The cardiomyocytes were then evenly seeded into a 12-well plate at a seeding density of 5 × 10 5 cells / well.

[0023] (2) Inosine treatment of primary neonatal mouse cardiomyocytes: 48 hours after cell seeding, primary cardiomyocytes were treated with complete culture medium containing 100 μM inosine (MCE, HY-N0092) or its control solvent DMSO for 24 hours.

[0024] (3) Cysteine ​​deprivation-induced ferroptosis: The culture medium of primary cardiomyocytes treated with control solvent was replaced with complete culture medium containing 10% FBS (normal control group) or cystine-free culture medium (model group), and the culture medium of primary cardiomyocytes treated with inosine was replaced with cystine-free culture medium containing 10% FBS (inosine-treated group) and cultured for 48 h.

[0025] (4) Observe and record cell morphology under a light microscope.

[0026] (5) If Figure 1 As shown in the figure, under the cystine-free [Cystine (-)+DMSO] culture condition, the cardiomyocytes showed obvious shrinkage, decreased in size, and cell death after 48 hours compared with the normal control group [Cystine (+)+DMSO]. However, the morphology of cardiomyocytes after inosine treatment [Cystine (-)+Inosine] did not change significantly compared with the normal group.

[0027] Example 2 (1) Isolation and culture of primary cardiomyocytes from suckling mice: The hearts of suckling mice within 1 day of birth were cut out with sterile ophthalmic scissors, washed once with PBS, chopped, and digested with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37°C for 10 min each time, for a total of 5 times. After each digestion, the supernatant was transferred to a high-glucose DMEM solution containing 20% ​​FBS and neutralized in a 1:1 ratio. After digestion, the resulting solution containing cardiomyocytes was filtered with a 70 μm filter and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended in a high-glucose DMEM solution containing 10% FBS. The cells were then transferred to a 10 cm cell culture dish and allowed to adhere differentially in a cell culture incubator at 37°C for 90 min. The supernatant was collected, the cells were counted, and diluted with a high-glucose DMEM solution with 10% FBS. 0.05 mM Brdu was added to inhibit the proliferation of fibroblasts. The cardiomyocytes were then evenly inoculated into a 96-well plate at a seeding density of 5 × 10 4 cells / well.

[0028] (2) Inosine pretreatment of primary neonatal mouse cardiomyocytes: 48 hours after cell inoculation, primary cardiomyocytes were treated with complete culture medium containing 100 μM inosine or control solvent for 24 hours.

[0029] (3) Induction of ferroptosis by inosine treatment: The culture medium of cardiomyocytes treated with control solvent was replaced with complete culture medium containing 10% FBS + DMSO (normal control group) or cysteine-free culture medium + DMSO (model group), or cysteine-free culture medium + 1 μM Ferr-1 (positive control group). The culture medium of primary cardiomyocytes treated with inosine was replaced with cysteine-free culture medium containing 10% FBS + DMSO (inosine-treated group) and cultured for 48 h.

[0030] (4) Cell viability assessment: The CellTiter-Glo kit (Promega, G7572) was used to evaluate the survival of neonatal rat cardiomyocytes.

[0031] (5) If Figure 2 As shown in the data, under cystine-free [Cystine (-)+DMSO] culture conditions, the viability of primary cardiomyocytes in the model group was 22.97±2.76%, which was significantly reduced compared with normal culture [Cystine (+)+DMSO]. The addition of Fer-1 [Cystine (-)+Fer-1] increased the cardiomyocyte survival rate to 71.33±6.14%, rescuing the occurrence of cardiomyocyte ferroptosis. Inosine treatment [Cystine (-)+Inosine] increased the cardiomyocyte survival rate to 50.11±7.99%, indicating that inosine treatment can reduce the occurrence of cardiomyocyte death caused by cystine deprivation, suggesting that inosine has a direct regulatory effect on cardiomyocyte survival.

[0032] Example 3 (1) Isolation and culture of primary cardiomyocytes from suckling mice: The hearts of one-day-old suckling mice were cut with sterile ophthalmic scissors, washed with PBS, chopped, and digested with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37°C for 10 min each time, for a total of 5 times. After each digestion, the supernatant was transferred to a DMEM solution containing 20% ​​FBS and neutralized in a 1:1 ratio. After digestion, the resulting solution containing cardiomyocytes was filtered with a 70 μm filter and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended in a DMEM solution containing 10% FBS. The cells were then transferred to a 10 cm cell culture dish and allowed to adhere differentially in a cell culture incubator at 37°C for 90 min. The dish was then removed, the supernatant was collected, the cells were counted, and diluted with a DMEM solution containing 10% FBS. 0.05 mM Brdu was added to inhibit the proliferation of fibroblasts. The cells were then evenly plated into a 12-well plate at a seeding density of 5 × 10 5 cells / well.

[0033] (2) Inosine treatment of primary neonatal mouse cardiomyocytes: 48 hours after cell seeding, primary cardiomyocytes were treated with complete culture medium containing 100 μM inosine or control solvent for 24 hours.

[0034] (3) Cysteine ​​deprivation-induced ferroptosis: The culture medium of primary cardiomyocytes treated with control solvent was replaced with complete culture medium containing 10% FBS (normal control group) or cystine-free culture medium (model group), and the culture medium of primary cardiomyocytes treated with inosine was replaced with cystine-free culture medium containing 10% FBS (inosine-treated group) and cultured for 48 h.

[0035] (4) Detection of 4HNE protein expression: discard the culture medium, wash twice with pre-cooled PBS, add RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors and lyse on ice for 30 minutes, BCA protein quantification, add 5× loading, heat the denatured protein in a 98°C metal bath for 30 minutes. Then perform immunoblotting experiment, primary antibody Anti-4 Hydroxynonenal antibody [Abcam, ab46545 (1:1000)], secondary antibody goat anti-rabbit IgG H&L (HRP) [Abcam, ab205718 (1:10000)], detect the expression level of 4HNE protein, and evaluate the effect of inosine on lipid peroxides.

[0036] (5) If Figure 3As shown in the figure, after 36 hours of cystine deprivation, the expression of 4HNE in cardiomyocytes of the model group [Cystine (-)+DMSO] was significantly increased compared with that of the normal control group [Cystine (+)+DMSO], indicating that the content of intracellular lipid peroxides was significantly increased after cystine deprivation, while the expression of 4HNE in the inosine treatment group [Cystine (-)+Inosine] was significantly decreased compared with the model group, indicating that inosine treatment can significantly reduce the accumulation of lipid peroxides after cystine deprivation, and inosine treatment can inhibit ferroptosis of cardiomyocytes.

[0037] Example 4 (1) Isolation and culture of primary cardiomyocytes from suckling mice: The hearts of one-day-old suckling mice were cut with sterile ophthalmic scissors, washed with PBS, chopped, and digested with 0.05% trypsin + 0.05% type II collagenase on a shaker at 37°C for 10 min each time, for a total of 5 times. After each digestion, the supernatant was transferred to a high-glucose DMEM solution containing 20% ​​FBS and neutralized in a 1:1 ratio. After digestion, the resulting solution containing cardiomyocytes was filtered with a 70 μm filter and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were resuspended in a DMEM solution containing 10% FBS. The cells were then transferred to a 10 cm cell culture dish and allowed to adhere differentially in a cell culture incubator at 37°C for 90 min. The supernatant was collected, the cells were counted, and diluted with a DMEM solution containing 10% FBS. 0.05 mM Brdu was added to inhibit the proliferation of fibroblasts. The cardiomyocytes were then evenly seeded into a 6-well plate at a seeding density of 1 × 10 6 cells / well.

[0038] (2) Inosine pretreatment of primary neonatal mouse cardiomyocytes: 48 hours after cell inoculation, primary cardiomyocytes were treated with complete culture medium containing 100 μM inosine or control solvent for 24 hours.

[0039] (3) Induction of ferroptosis by inosine treatment: The culture medium of primary cardiomyocytes treated with control solvent was replaced with complete culture medium containing 10% FBS + DMSO (normal control group) or cystine-free culture medium + DMSO (model group), or cystine-free culture medium + 1 μM Fer-1 (positive control group). The culture medium of primary cardiomyocytes treated with inosine was replaced with cystine-free culture medium containing 10% FBS + DMSO (inosine-treated group) and cultured for 48 h.

[0040] (4) The cells were stained with 1 μM BODIPY 581 / 591 C11 (Invitrogen, D3861), incubated at 37°C for 30 min, trypsinized, neutralized, washed with PBS, and then the lipid peroxide content of each group was evaluated by flow cytometry.

[0041] (5) If Figure 4As shown in the figure, after 36 hours of cystine deprivation, flow cytometry analysis showed that the average fluorescence intensity of the FITC channel of the cardiomyocytes in the model group [Cystine (-)+DMSO] was significantly increased compared with that in the normal control group [Cystine (+)+DMSO], indicating that the intracellular lipid peroxide content was significantly increased after cystine deprivation, while the average fluorescence intensity of the FITC channel of the inosine treatment group [Cystine (-)+Inosine] was significantly decreased compared with the model group, indicating that inosine treatment can significantly reduce the accumulation of lipid peroxides after cystine deprivation, and inosine treatment can inhibit ferroptosis of cardiomyocytes.

[0042] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0043] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Application of inosine in the preparation of inhibitors of myocardial cell ferroptosis.

2. Application of inosine in the preparation of therapeutic drugs for myocardial cell ferroptosis-related diseases.

3. The use according to claim 2, characterized in that: The therapeutic drug comprises inosine and a pharmaceutically acceptable carrier, excipient or solvent.

4. The use according to claim 3, characterized in that: The therapeutic drug is an oral preparation or an injectable preparation.

5. The use of reagents that can promote the synthesis of inosine in the body in the preparation of inhibitors of myocardial cell ferroptosis.

6. The use of reagents that can promote the synthesis of inosine in the body in the preparation of therapeutic drugs for myocardial cell ferroptosis-related diseases.

7. The use according to claim 5 or 6, characterized in that: The agent capable of promoting the in vivo synthesis of inosine is selected from an adeno-associated virus vector inserted with an adenosine deaminase gene or an extracellular 5'-nucleotidase gene.

8. The use according to claim 6, characterized in that: The therapeutic drug comprises an agent capable of promoting the synthesis of inosine in vivo and a pharmaceutically acceptable carrier, excipient or solvent.

9. The use according to claim 8, characterized in that: The therapeutic drug is an injection preparation.

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