Application of interferon gene stimulating protein in myocardial ischemia reperfusion injury

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of interferon gene stimulating protein in myocardial ischemia reperfusion injury. The invention discloses a mechanism that interferon gene stimulating protein triggers ferroptosis in myocardial cells and aggravates myocardial ischemia-reperfusion injury. In other words, MI / R stimulates accumulation of cytoplasm double-stranded DNA, and a cyclized guanylate-adenylate synthetase-cyclic guanylate-adenylate-STING signal channel is activated. Activated STING is directly combined with glutathione peroxidase 4 to induce autophagy degradation of GPX4, oxidative stress is initiated, and finally ferroptosis is triggered in myocardial cells. The specific targeting of STING can alleviate ferroptosis and heart injury induced by ischemia reperfusion. The invention not only discloses a new molecular mechanism behind GPX4 related cell death, but also determines STING as a promising therapeutic target for treating MI / R injury, and has good potential practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to the application of interferon gene-stimulating protein in myocardial ischemia-reperfusion injury. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Ischemic heart disease remains the leading cause of morbidity and mortality worldwide. Although timely coronary intervention reperfusion therapy can effectively rescue ischemic myocardium, myocardial ischemia-reperfusion (MI / R) injury is still an important factor leading to death in a considerable proportion of patients. So far, despite decades of in-depth research, no drug specifically targeting myocardial ischemia-reperfusion injury has entered the clinical application stage. At present, there is an urgent medical need to comprehensively and deeply understand the complex mechanisms regulating cardiomyocyte death. Mastering these mechanisms is crucial because it can prompt people to discover new and innovative therapeutic targets, which is extremely critical for effectively dealing with related pathological conditions.

[0004] Cell death, especially cardiomyocyte death, is a key aspect in the pathophysiology of cardiovascular diseases. Among various forms of cell death, ferroptosis, as an iron-dependent form of cell death characterized by intense lipid peroxidation, has become a key factor in the pathophysiology of myocardial ischemia-reperfusion (MI / R) injury. Ferroptosis is regulated by iron, lipid, amino acid, and glutathione metabolism, and is closely related to various heart diseases. This makes intervening in ferroptosis a promising therapeutic method for treating myocardial ischemia-reperfusion injury, which may revolutionize the treatment strategy for this common and life-threatening disease. Glutathione peroxidase 4 (GPX4), which has the function of converting lipid peroxides into less harmful lipid alcohols, has become a key regulator for inhibiting ferroptosis. Previous studies have shown that ferroptosis induced by myocardial ischemia-reperfusion is accompanied by the inhibition of glutathione peroxidase 4 (GPX4). During myocardial ischemia-reperfusion, the decrease in GPX4 level occurs simultaneously with the initiation of ferroptosis. On the contrary, increasing the GPX4 level can effectively reduce myocardial injury and improve cardiac function. However, the precise regulation of GPX4 protein level and its potential degradation mechanism remain unclear. Therefore, discovering related factors regulating GPX4 may provide an attractive therapeutic target for modulating ferroptosis occurring during myocardial ischemia-reperfusion. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide the application of stimulator of interferon genes (STING) in myocardial ischemia-reperfusion injury. Specifically, through research, the present invention finds that ischemia-reperfusion (I / R)-induced mitochondrial damage leads to the release of double-stranded DNA (dsDNA), which is then recognized by cyclic GMP-AMP synthase (cGAS), and then the second messenger cyclic GMP-AMP (cGAMP) is produced and the stimulator of interferon genes (STING) is activated. STING can directly trigger ferroptosis of cardiomyocytes by interacting with glutathione peroxidase 4 (GPX4). Once STING binds to GPX4, it promotes the enhancement of autophagy, which then leads to the degradation of GPX4, thereby promoting the process of ferroptosis of cardiomyocytes. We also evaluated two effective treatment strategies for myocardial ischemia-reperfusion injury, including overexpression of GPX4 mediated by adeno-associated virus (AAV) and the use of STING antagonists. Based on our research, the obtained results provide a new perspective on the mechanism of ferroptosis and identify STING as a potential therapeutic target, which is expected to be used to prevent myocardial ischemia-reperfusion injury, improve the prognosis of patients and reduce the mortality associated with ischemic heart disease. Based on the above research results, the present invention is thus completed.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, there is provided the application of a reagent for detecting the expression level of STING and its related biomolecules in the preparation of a detection product for myocardial ischemia-reperfusion injury.

[0008] Furthermore, the detection product for myocardial ischemia-reperfusion injury can be used for screening, (assistant) diagnosis, monitoring or prognosis evaluation of myocardial ischemia-reperfusion injury.

[0009] Specifically, during myocardial ischemia-reperfusion, the double-stranded DNA (dsDNA)-cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signal accumulates, accompanied by a relatively high incidence of ferroptosis in cardiomyocytes. Specific deletion of cgas or Sting in cardiomyocytes can inhibit oxidative stress and has been proven to be able to reduce ferroptosis and ischemia-reperfusion injury. On the contrary, activation of STING exacerbates ferroptosis and ischemia-reperfusion injury. Mechanistically, STING directly acts on glutathione peroxidase 4 (GPX4) and promotes the fusion of autophagosomes with lysosomes to degrade GPX4 in an autophagic manner. This STING-GPX4 axis promotes ferroptosis of cardiomyocytes and forms a positive feedback loop. Therefore, the STING and its related biomolecules can be used for screening, (assistant) diagnosis, monitoring or prognosis of myocardial ischemia-reperfusion injury.

[0010] Among them, the related biomolecules of STING include any one or more of double-stranded DNA, cGAS, cGAMP, and GPX4.

[0011] The substances for detecting the expression levels of STING and its related biomolecules may include reagents for detecting the expression levels of the genes encoding STING and its related biomolecules based on real-time fluorescence quantitative PCR, in situ hybridization, gene chips, and gene sequencing, and / or reagents for detecting the protein (enzyme) expression levels of STING and its related biomolecules based on immunoassay methods.

[0012] In the present invention, the product may be primers, probes, (gene or protein) chips, detection kits, detection devices, etc., which are not specifically limited herein.

[0013] In the second aspect of the present invention, a system for detecting myocardial ischemia-reperfusion injury is provided, and the system includes:

[0014] An acquisition unit configured to acquire the expression level situation of STING and its related biomolecules of a subject;

[0015] An analysis unit configured to analyze and judge the disease condition of the subject according to the expression level situation of STING and its related biomolecules of the subject obtained by the acquisition unit.

[0016] Among them, the related biomolecules of STING include any one or more of double-stranded DNA, cGAS, cGAMP, and GPX4.

[0017] Among them, the sample to be tested of the subject may be a myocardial cell sample of the subject.

[0018] The detection of myocardial ischemia-reperfusion injury may specifically be the screening, (auxiliary) diagnosis, monitoring, or prognosis of myocardial ischemia-reperfusion injury.

[0019] In the third aspect of the present invention, the application of STING and its related biomolecules as targets in the preparation and / or screening of drugs for myocardial ischemia-reperfusion injury is provided.

[0020] Furthermore, based on the effects of a candidate drug on STING and its related biomolecules before and after use, it is determined whether the candidate drug is used for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0021] Furthermore, the method for screening drugs for preventing and / or treating myocardial ischemia-reperfusion injury includes:

[0022] (I) Treating a system expressing and / or containing STING and its related biomolecules with a candidate substance; setting a control without treating with the candidate substance;

[0023] (II) After completing step (I), detect the expression levels of STING and its related biomolecules in the system; compared with the control, if the expression levels of STING and its related biomolecules in the system treated with the candidate substance change significantly, the candidate substance can be used as a candidate drug for preventing or treating myocardial ischemia-reperfusion injury.

[0024] Among them, the related biomolecules of STING include GPX4.

[0025] More specifically, the specific method of step (II) is: if the expression level of STING is significantly down-regulated and / or the expression level of GPX4 is significantly up-regulated in the system treated with the candidate substance, the candidate substance can be used as a candidate drug for preventing and / or treating myocardial ischemia-reperfusion injury;

[0026] The system can be a solution system, a cell system (such as cardiomyocytes), a tissue system, an organ system or an animal system, and no specific limitation is made here.

[0027] In the fourth aspect of the present invention, there is provided an application of a substance that inhibits the expression of STING or reduces its activity in any one or more of the following:

[0028] (a) Inhibiting the degradation of GPX4 or preparing a product for inhibiting the degradation of GPX4;

[0029] (b) Inhibiting ferroptosis or preparing a product for inhibiting ferroptosis;

[0030] (c) A product for preventing and / or treating myocardial ischemia-reperfusion injury.

[0031] Among them, the substance that inhibits the expression of STING or reduces its activity includes but is not limited to RNA interference molecules or antisense oligonucleotides against STING, small molecule inhibitors (such as H-151, CAS: 941987-60-6), siRNA, shRNA, substances for implementing lentiviral infection or gene knockout, etc.

[0032] Among them, in (a)-(c) above, the product can act on cardiomyocytes, myocardial tissue and / or the heart.

[0033] The product can be a drug or an experimental reagent for non-medical use, and the experimental reagent can be used for basic research. For example, the product can be used to regulate ferroptosis of cardiomyocytes in vivo (in vitro), etc., so as to prepare a biological model related to cardiomyocytes, thereby providing a basis for the mechanism research of diseases such as myocardial ischemia-reperfusion injury.

[0034] According to the present invention, when the product is a drug, the drug also includes at least one pharmaceutically inactive ingredient.

[0035] The drug can be formulated into oral preparations, external preparations, suppositories, and sterile injection solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc. according to conventional practices.

[0036] The drug may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a conditioner, a surfactant, a dispersant or an antifoaming agent.

[0037] Furthermore, the delivery vehicle of the pharmaceutically acceptable carrier may be liposomes, biocompatible polymers (including natural polymers and synthetic polymers), lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial virus envelopes, inorganic (including metal) particles, and bacterial, phage, cosmid or plasmid vectors, etc., so as to broaden its application scope.

[0038] The drug of the present invention can be administered into the body by known methods. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Optionally, it can be administered via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the administration route, the administration mode, and so on.

[0039] The subjects to which the drug is administered can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys and chimpanzees.

[0040] In a fifth aspect of the present invention, there is provided the use of a substance that inhibits STING expression or reduces its activity in combination with a substance that inhibits the degradation of GPX4 in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury.

[0041] Among them, the substance that inhibits STING expression or reduces its activity can be a STING inhibitor, specifically H-151. The substance that inhibits the degradation of GPX4 can be any one or more of lysosome inhibitors or autophagy inhibitors. Among them, the lysosome inhibitors include chloroquine and ammonium chloride, and the autophagy inhibitors include bafilomycin A1 (BafA-1), LY294002, 3-methyladenine (3-MA), and wortmannin. The substance that inhibits the degradation of GPX4 is preferably bafilomycin A1. The combination of the two (the mass ratio of H-151 to bafilomycin A1 is 3-8:1-5, preferably 5:3) can significantly improve the necrotic area after myocardial ischemia-reperfusion, and the cardiac function (ejection fraction and shortening fraction levels) is significantly better. The fibrotic area is significantly reduced, and the increase in the level of 4-hydroxy-2-nonenal (4-HNE) caused by I / R is alleviated, and the effect is better than that of each of them used alone, producing a good synergistic effect against myocardial ischemia-reperfusion injury.

[0042] In a sixth aspect of the present invention, there is provided a method for preventing and / or treating myocardial ischemia-reperfusion injury, the method comprising administering to a subject the above-mentioned substance that inhibits STING expression or reduces its activity and / or the corresponding product or drug.

[0043] In the present invention, the myocardial ischemia-reperfusion injury includes ischemic heart disease mediated by myocardial ischemia-reperfusion injury, which is not specifically limited herein.

[0044] The beneficial technical effects of the above one or more technical solutions:

[0045] The above technical solutions for the first time reveal the mechanism by which stimulator of interferon genes (STING) triggers ferroptosis in cardiomyocytes (CMs) and exacerbates myocardial ischemia-reperfusion (MI / R) injury. That is, MI / R stimulates the accumulation of cytoplasmic double-stranded DNA (dsDNA), activates the cyclic guanosine monophosphate-adenylate synthase (cGAS)-cyclic guanosine monophosphate-adenylate (cGAMP)-STING signaling pathway. The activated STING directly binds to glutathione peroxidase 4 (GPX4), induces autophagic degradation of GPX4, triggers oxidative stress, and finally triggers ferroptosis in cardiomyocytes. Specifically targeting STING may reduce ischemia-reperfusion (I / R)-induced ferroptosis and cardiac injury. These results indicate that the STING-GPX4 axis plays a key role in cardiomyocyte ferroptosis. This not only reveals a new molecular mechanism behind GPX4-related cell death, but also identifies STING as a promising therapeutic target for treating MI / R injury, and thus has good potential practical application value. Brief Description of the Drawings

[0046] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments and descriptions thereof of the invention are used to explain the invention and do not unduly limit the invention.

[0047] Figure 1 In the embodiment of the present invention, ischemia-reperfusion (I / R) triggers the upregulation of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) in cardiomyocytes

[0048] Male mice at 8 weeks of age were surgically operated and euthanized 24 hours after ischemia-reperfusion (I / R) or after sham operation.

[0049] (a) shows the surgical flow chart of the mouse I / R model. (b) Schematic diagram of the mouse I / R injury model. (c) Double immunofluorescence analysis was performed on the same heart section of the infarct border zone of wild-type (WT) mice after I / R or sham operation to detect double-stranded DNA (dsDNA) and cGAS. The positive reactions of the tissue sections are shown in green (dsDNA) and red (cGAS), respectively. The co-localized positive reactions are shown in yellow. Scale bar = 20 μm. (d-e) Protein immunoblot analysis and quantification of cGAS and STING in wild-type (WT) cells isolated from the infarct border zone after I / R or sham operation (n = 6). (f-g) Protein immunoblot analysis and quantification of cGAS and STING in various cell types isolated from the infarct border zone of cgas knockout (cgas- / -), Sting knockout (Sting- / -) or wild-type (WT) mice after I / R or sham operation (n = 6). (h) Double immunofluorescence analysis was performed on the same heart section of the infarct border zone of wild-type (WT) mice after I / R or sham operation to detect cardiomyocyte (CMs) markers and STING. The positive reactions of the tissue sections are shown in green (STING) and red (troponin), respectively. The co-localized positive reactions are shown in yellow. Scale bar = 20 μm. (i) Schematic diagram of the operation flow chart of cardiomyocyte hypoxia-reoxygenation (A / R). (j) Double immunofluorescence analysis was performed on primary mouse cardiomyocytes (MPCs) after hypoxia-reoxygenation (A / R) to detect dsDNA and mitochondria (labeled with mitochondrial tracer Mito-Tracker). The positive reactions are shown in green (dsDNA) and red (Mito-Tracker), respectively. The co-localized positive reactions are shown in yellow. Arrows indicate the released dsDNA. Scale bar = 20 μm.

[0050] Figure 2In the embodiments of the present invention, knocking out cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) can protect the myocardium from ischemia-reperfusion (I / R) injury

[0051] (a) Schematic diagram of the structure of cardiomyocyte-specific (Myh6-iCre) conditional knockout mice of cgas or Sting. (b-e) Effects of cardiomyocyte-specific knockout of cgas (cgas-CKO) on myocardial infarction area, fractional shortening (FS), ejection fraction (EF), and fibrotic area after I / R or sham operation: (b) Myocardial infarction area (percentage of the area at risk (AAR)) and representative tissue sections (n = 6); (c) Echocardiography and measured ejection fraction (EF%) and fractional shortening (FS%) (n = 7); (d) Masson staining and measured percentage of fibrotic area (n = 7), scale bar = 1 mm; (e) Western blot analysis and quantification of cGAS in cardiomyocytes (CMs) isolated from the border region of the cardiac infarct (n = 5). (f-i) Effects of cardiomyocyte-specific knockout of Sting (Sting-CKO) on myocardial infarction area, fractional shortening (FS), ejection fraction (EF), and fibrotic area after I / R: (f) Myocardial infarction area (percentage of the area at risk (AAR)) and representative tissue sections (n = 6); (g) Echocardiography and measured ejection fraction (EF%) and fractional shortening (FS%) (n = 7); (h) Masson staining and measured percentage of fibrotic area (n = 6), scale bar = 1 mm; (i) Western blot analysis and quantification of STING in cardiomyocytes (CMs) isolated from the border region of the cardiac infarct (n = 6). Data are expressed as mean ± standard error of the mean (Mean+SEM), **P<0.01, ***P<0.001, ****P<0.0001. AAR: Area at risk; IF: Infarct area; cgas-CKO: cgasfl / fl Myh6iCre; Sting-CKO: Stingfl / fl Myh6iCre; FS: Fractional shortening; EF: Ejection fraction; KO: Knockout.

[0052] Figure 3 In the embodiments of the present invention, STING (stimulator of interferon genes) exacerbates myocardial ferroptosis by regulating oxidative stress injury

[0053] (a) Heart regions for RNA sequencing after ischemia reperfusion (I / R). (b) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment bar graph of RNA sequencing. (c) Statistical chart of immunofluorescence images of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays on heart sections of infarct border regions of cgas cardiomyocyte-specific knockout (cgas-CKO) or Sting cardiomyocyte-specific knockout (Sting-CKO) mice and their control mice after I / R (n = 6). (d) Gene Ontology (GO) enrichment scatter plot of RNA sequencing. (e) Malondialdehyde (MDA) assay for detecting lipid peroxidation (n = 6). (f) Live cell immunofluorescence imaging analysis for detecting reactive oxygen species (ROS) in mouse primary cardiomyocytes (MPCs). Scale bar = 20 μm. (g-h) Western blot analysis and quantification of acyl-CoA synthetase long-chain family member 4 (ACSL4), transferrin receptor (TFR), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in Stingfl / fl or Sting-CKO cardiomyocytes (CMs) after I / R (n = 6). (i-j) Western blot analysis and quantification of ACSL4, TFR, SLC7A11, and GPX4 in primary cardiomyocytes (MPCs) of Stingfl / fl or Sting-CKO mice under hypoxia-reoxygenation (A / R) or normoxia conditions (n = 6). Data are represented as mean ± standard error of the mean (Mean + SEM), *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0054] Figure 4 Targeting of stimulator of interferon genes (STING) by glutathione peroxidase 4 (GPX4) in the examples of the present invention

[0055] (a) Ferroptosis pathway enrichment shown by the Kyoto Encyclopedia of Genes and Genomes (KEGG). (b) Silver staining and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis using a STING protein affinity antibody for tandem affinity purification in mouse primary cardiomyocytes (MPCs) after hypoxia-reoxygenation (A / R). (c) Images of ten potential contact modes shown by the molecular docking results between STING (Protein Data Bank (PDB) ID: 4F5W) and GPX4 (PDB ID: 5L71). (d) Co-immunoprecipitation (Co-IP) experiments on mouse primary cardiomyocytes (MPCs) to detect whether endogenous GPX4 interacts with STING and whether this binding is affected by short-time point stimulation with cyclic guanosine monophosphate-adenylate (cGAMP). Immunoblotting (IB): STING, immunoprecipitation (IP): GPX4. (e) Co-IP experiments on mouse primary cardiomyocytes (MPCs) to detect whether endogenous STING interacts with GPX4 and whether this binding is affected by short-time point stimulation with cGAMP. Immunoblotting (IB): GPX4, immunoprecipitation (IP): STING. (f) Co-IP experiments on human cervical cancer cells (HeLa) co-transfected with Flag-tagged GPX4 (Flag-GPX4) and Myc-tagged STING (Myc-STING) to detect whether STING interacts with GPX4. Immunoblotting (IB): Myc, immunoprecipitation (IP): Flag. (g) Co-IP experiments on HeLa cells co-transfected with Flag-GPX4 and Myc-STING to detect whether GPX4 interacts with STING. Immunoblotting (IB): Flag, immunoprecipitation (IP): Myc. (h) Co-IP experiments on HeLa cells co-transfected with Flag-GPX4 and Myc-STING under the condition of delivering erastin or cGAMP to detect whether the interaction between GPX4 and STING is affected by long-time action of cGAMP or erastin. Immunoblotting (IB): Flag, immunoprecipitation (IP): Myc. (i) Double immunofluorescence analysis on HeLa cells co-transfected with Myc-STING and Flag-GPX4 under the condition of adding cGAMP or erastin to detect STING and GPX4 and their co-localization. The positive reaction of the Myc label is shown in red, and the positive reaction of the Flag label is shown in green. The positive reaction of co-localization is shown in yellow. Scale bar = 20 μm.(j) Double immunofluorescence analysis of the same heart sections from the infarct border regions of Stingfl / fl or Sting-CKO mice was performed to detect GPX4 and STING. Positive reactions in tissue sections are shown in green (GPX4) and red (STING), respectively. Co-localized positive reactions are shown in yellow. Scale bar = 40 μm. LC: liquid chromatography; MS: mass spectrometry; PDB: protein data bank; Co-IP: co-immunoprecipitation; IN: input sample; IB: immunoblotting; IP: immunoprecipitation; Flag-GPX4: Flag-tagged GPX4 plasmid; Myc-STING: Myc-tagged STING plasmid.

[0056] Figure 5This is a representative image of the molecular docking results showing the direct interaction between STING (stimulator of interferon genes) and glutathione peroxidase 4 (GPX4) at the N146 amino acid residue of GPX4 and the T267 amino acid residue of STING in the embodiments of the present invention, demonstrating potential contact sites between GPX4 and STING. (b) Co-immunoprecipitation (Co-IP) experiments were performed on human cervical cancer cells (HeLa) co-transfected with Flag-tagged GPX4 (Flag-GPX4) or Flag-GPX4 point mutants (Flag-GPX4[ΔG126], Flag-GPX4[ΔR127], or Flag-GPX4[ΔN146]) and Myc-tagged STING (Myc-STING) to detect potential contact sites between GPX4 and STING. Immunoblotting (IB): Myc, Immunoprecipitation (IP): Flag. (c) Co-immunoprecipitation (Co-IP) experiments were performed on HeLa cells co-transfected with Flag-GPX4 and Myc-STING or Myc-STING point mutants (Myc-STING[ΔY167], Myc-STING[ΔE260], Myc-STING[ΔY245], Myc-STING[ΔQ266], or Myc-STING[ΔT267]) to detect potential contact sites between GPX4 and STING. Immunoblotting (IB): Myc, Immunoprecipitation (IP): Flag. (d) Double immunofluorescence analysis was performed on HeLa cells co-transfected with Myc-STING and Flag-GPX4, Flag-GPX4 and Myc-STING[ΔT267], or Myc-STING and Flag-GPX4[ΔN146] to detect STING and GPX4 and their co-localization. The positive reaction of the Myc label is shown in red, and the positive reaction of the Flag label is shown in green. The positive reaction of co-localization is shown in yellow. Scale bar = 20 μm. (e) Double immunofluorescence analysis was performed on HL-1 cells co-transfected with Myc-STING and Flag-GPX4 or Myc-STING and Flag-GPX4[ΔN146] with or without cyclic guanosine monophosphate-adenylate (cGAMP) added to detect STING and GPX4 and their co-localization. The positive reaction of the Myc label is shown in green, and the positive reaction of the Flag label is shown in red. The positive reaction of co-localization is shown in yellow. Scale bar = 5 μm.G: Glycine; R: Arginine; N: Asparagine; Y: Tyrosine; E: Glutamic acid; Q: Glutamine; T: Threonine.

[0057] Figure 6 In the embodiment of the present invention, STING (Stimulator of Interferon Genes) promotes ferroptosis through autophagy-lysosome-mediated degradation of glutathione peroxidase 4 (GPX4).

[0058] (a) Protein immunoblot analysis and quantification to detect the effect of ferroptosis inhibitor-1 (Fer-1) or cyclic GMP-AMP (cGAMP) on the expression of GPX4 in mouse primary cardiomyocytes (MPCs) (n = 5). (b) Protein immunoblot analysis and quantification to detect the effect of erastin, cGAMP or H-151 on the degradation of GPX4 in MPCs (n = 5). (c) Protein immunoblot analysis and quantification to detect cGAMP-induced degradation of GPX4 in MPCs and the inhibitory effect of MG-132, ammonium chloride (NH4Cl), chloroquine and calpain inhibitor on it (n = 5). (d) Protein immunoblot analysis and quantification to detect cGAMP-induced degradation of GPX4 in MPCs and the inhibitory effect of bafilomycin A1 (Baf A-1), LY294002, 3-methyladenine (3-MA) and wortmannin on it (n = 5). (e-g) Immunofluorescence analysis to show the autophagic flux of Stingfl / fl or cardiomyocyte-specific knockout of Sting (Sting-CKO) MPCs with or without hypoxia-reoxygenation (A / R) using mRFP-GFP-LC3. Positive autophagic reactions are shown as red dots. Scale bar = 10 μm. (h) Triple immunofluorescence analysis to detect GPX4, STING and microtubule-associated protein 1A / 1B light chain 3B (LC3B) in Stingfl / fl or Sting-CKO MPCs. Positive co-localization reactions are shown as yellow (co-localization of LC3B and STING) or pink (triple co-localization). Scale bar = 20 μm. (i) Triple immunofluorescence analysis to detect GPX4, STING and lysosome-associated membrane protein 2B (LAMP2B) in MPCs with or without A / R. Positive co-localization reactions are shown as yellow (co-localization of GPX4 and STING) or pink (triple co-localization). Scale bar = 20 μm. Data are expressed as mean ± standard error of the mean (Mean+SEM), NS indicates no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001, P < 0.001. Baf A-1: bafilomycin A1; LAMP2B: lysosome-associated membrane protein 2B.

[0059] Figure 7 In the embodiment of the present invention, adeno-associated virus (AAV)-mediated glutathione peroxidase 4 (GPX4) therapy can protect cardiac function against severe ischemia-reperfusion (I / R) injury triggered by the activation of STING (stimulator of interferon genes).

[0060] Effect of AAV-cTNT-GPX4 on the treatment of cardiac dysfunction in C57BL / 6J mice administered 5,6-dimethylxanthenone-4-acetic acid (DMXAA) or dimethyl sulfoxide (DMSO) after I / R: (a) Schematic diagram depicting the time course of I / R-induced cardiac dysfunction treated with AAV or DMXAA. (b) Western blot analysis to verify successful overexpression of GPX4 in cardiomyocytes (CMs). (c-d) Infarct size (percentage of area at risk (AAR)) and representative tissue sections (n = 6). (e-f) Echocardiography and measured ejection fraction (EF%) and fractional shortening (FS%) (n = 6). (g) Double immunofluorescence analysis of GPX4 and 4-hydroxynonenal (4-HNE) on the same cardiac sections of the infarct border zone. Scale bar = 20 μm. (h) Immunofluorescence imaging analysis to detect reactive oxygen species (ROS) in cardiomyocytes of mice infected with AAV-cTNT-GPX4 or AAV-control virus in the presence of DMSO or DMXAA after exposure to hypoxia-reoxygenation (A / R). Scale bar = 20 μm. Data are expressed as mean ± standard error of the mean (Mean+SEM), NS indicates no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001, #P < 0.05, P < 0.001. AAV: adeno-associated virus; 4-HNE: 4-hydroxynonenal; cTNT-GPX4: AAV-Mus-GPX4-cTNT-C-GFP; DMXAA: Vadimezan.

[0061] Figure 8 In the embodiment of the present invention, STING (stimulator of interferon genes) is a potential therapeutic target for inhibiting cardiac dysfunction after ischemia-reperfusion (I / R).

[0062] (a) i. Schematic diagram of the time course of I / R-induced cardiac dysfunction treated with H-151 or dimethyl sulfoxide (DMSO). ii. Representative photographs of the heart after I / R with the addition of DMSO or H-151. (b-c) Infarct size (percentage of the area at risk (AAR)) and representative tissue sections after the use of H-151 (n = 6). (d-e) Echocardiography and measured ejection fraction (EF%) and fractional shortening (FS%) after the use of H-151 (n = 6). (f) Double immunofluorescence analysis of glutathione peroxidase 4 (GPX4) and 4-hydroxynonenal (4-HNE) on the same cardiac section of the infarct border zone. Scale bar = 200 μm. (g) Immunofluorescence analysis of GPX4 and STING in mouse cardiomyocytes (CMs) with the addition of DMSO or H-151 after hypoxia-reoxygenation (A / R). Scale bar = 20 μm. (h) Immunofluorescence analysis of autophagic flux shown by mRFP-GFP-LC3 in mouse cardiomyocytes with the addition of DMSO or H-151 after A / R. Positive reactions of autophagy are shown as red dots. Scale bar = 20 μm. (i) Immunofluorescence imaging analysis of reactive oxygen species (ROS) in mouse cardiomyocytes with the addition of DMSO or H-151 after A / R. Scale bar = 20 μm. (j) Statistical chart of infarct size (% of AAR) in C57BL / 6J mice after different drug treatments after I / R (n = 6). (k) EF% and FS% of cardiac function measurement in C57BL / 6J mice after different drug treatments after I / R (n = 6). (l) Statistical chart of 4-HNE fluorescence intensity in the same cardiac section of the border zone in C57BL / 6J mice after different drug treatments after I / R (n = 6). Data are expressed as mean ± standard error of the mean (Mean + SEM), NS indicates no significant difference, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed Description

[0063] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs.

[0064] It should be noted that the terms used herein are merely for describing the specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0065] The present invention will be further described below in connection with specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0066] The present invention will be further explained and illustrated below by way of examples, but this does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions indicated in the following examples are usually carried out under conventional conditions.

[0067] Example

[0068] Materials and Methods

[0069] Ethical Statement: All animal experiments were strictly conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH), USA. The Animal Experiment Management Committee of Qilu Hospital of Shandong University (approval number: DWLL-2021-206) gave full approval to ensure that the operations were in line with ethical norms and responsibilities were clearly defined throughout the research process. This clinical study was conducted in accordance with the principles stipulated in the Declaration of Helsinki and was approved by the Ethics Committee of Qilu Hospital of Shandong University (approval number: KYLL-2022(ZM)-1344). All patients participating in the study have signed the informed consent form.

[0070] Ischemia / Reperfusion (I / R) Animal Model: To establish an I / R animal model, 8-week-old male C57BL / 6J mice were selected. These mice were securely fixed on a mouse fixing plate with a constant temperature of 37°C. Before the operation, the chest area of the mice was thoroughly disinfected with iodophor. The mice were anesthetized by inhalation with 3% isoflurane at a flow rate of 1 liter per minute. Using sterile scissors, a surgical incision about 2 cm long was made in the chest at the most obvious part of the mouse's heartbeat. A 4-0 purse-string suture was placed, and then the thoracic cavity above the heart was dissected through the fourth intercostal space with curved forceps. The chest was gently pressed, and the heart was carefully extruded from the thoracic cavity and placed in the suture opening, ensuring the least amount of residual air in the thoracic cavity. Then, a surgical slipknot was made around the left anterior descending coronary artery with a 6-0 surgical suture, and one end of the suture was left outside the thoracic cavity. Immediately, the heart was returned to the thoracic cavity, the excess air was discharged, and the epidermal incision was sutured. After the operation, the mice were transferred to the anesthesia recovery room. After 30 minutes, the slipknot for heart reperfusion was loosened, and then the mice were returned to the breeding cage for further care.

[0071] Subsequently, the heart was removed and stained to accurately determine the extent of myocardial necrosis, expressed as the percentage of the non-perfused area at risk (AAR). The ischemic area containing viable tissue was stained a distinct red with 2,3,5-triphenyltetrazolium chloride, while the non-ischemic area was clearly labeled blue with Evans blue. Then, the heart was frozen at -80 °C for 10 minutes and sliced (5 - 6 slices per heart). The infarct area, area at risk (AAR), and left ventricular (LV) area were measured using ImageJ software from the National Institutes of Health (NIH). The infarct size was calculated as the ratio of the infarct area to the area at risk (AAR). These slices were photographed with a gross imaging microscope (Leica M205 FA), and the images were subsequently analyzed using NIH Image software.

[0072] Echocardiography: Twenty-four hours after model establishment, transthoracic echocardiography was used to evaluate the cardiac structure and function of the mice. The VisualSonic VeVo 2100 imaging system from Toronto, Canada, was used for this evaluation. Before starting the examination, the mice were anesthetized by inhaling 2% isoflurane. Subsequently, the mice were placed on a heated platform maintained at 37 ± 1 °C and securely connected to an electrocardiogram (ECG) for continuous monitoring. By applying M-mode echocardiography, the left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs) on the parasternal long axis were carefully recorded. Finally, the left ventricular ejection fraction and fractional shortening were automatically calculated for precise evaluation.

[0073] Isolation of different types of myocardial cell populations: Twenty-four hours after myocardial ischemia-reperfusion (MI / R) in mice, different types of myocardial cell populations were isolated from the infarcted hearts of the mice. This operation was carried out according to the existing experimental protocol. Standardized techniques were used to isolate cardiomyocytes (CMs). To isolate cardiac fibroblasts (CFs) and macrophages, a skeletal muscle dissociation kit (Miltenyi Biotec, Shanghai, China) was used. To separate macrophages from cardiac fibroblasts, magnetic beads coated with anti-F4 / 80 (product number: 130-110-443; Miltenyi Biotec) were used strictly according to the manufacturer's instructions. Subsequently, the purified cells were collected by centrifugation at 300 × g for 5 minutes at 4 °C in preparation for subsequent protein extraction.

[0074] Immunofluorescence staining, Masson's trichrome (MT) staining, and 4-hydroxynonenal (4-HNE) staining: The infarct area was defined as the part between the suture and the apex. On the short-axis cross-section, the infarct border zone was carefully demarcated as the marginal zone differentiating infarcted tissue from adjacent non-infarcted myocardium. Using a microtome (RM2235; Leica Microsystems, Mannheim, Germany), cardiac tissue 1 mm below the ligated site was accurately cut into 4-μm-thick transverse sections and arranged along the horizontal long axis. Serial sections were used for the MT staining procedure to evaluate myocardial fibrosis. For immunofluorescence staining of double-stranded DNA (dsDNA), cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), stimulator of interferon genes (STING), glutathione peroxidase 4 (GPX4), and 4-HNE, the sections were first dewaxed. Then antigen retrieval was performed using a dedicated kit (C1034; Solarbio, Beijing, China). Subsequently, the sections were immersed in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 (GC204003; Sevier Bio, Wuhan, China) for 10 minutes to increase their permeability. Next, at room temperature (23 - 27 °C), the sections were incubated with PBS solution containing 5% normal goat serum (G1208; Sevier Bio, Wuhan, China) for 60 minutes to block non-specific binding sites. Then, specific antibodies against dsDNA, cGAS, STING, GPX4, or 4-HNE were added, and the sections were incubated overnight at 4 °C. After that, the sections were washed three times with PBS solution to remove unbound antibodies. Subsequently, in the dark at 37 °C, the sections were incubated with secondary antibodies Alexa Fluor 594 (ab150120; Abcam, Cambridge, Massachusetts, USA) and Alexa Fluor 488 (ab150081; Abcam, USA) (diluted at a ratio of 1:200) for 1 hour to observe the specific binding of the primary antibody. Finally, the cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, ab104139; Abcam, UK) for clear identification of cell localization.

[0075] Cell culture: Human embryonic kidney 293T cells (HEK293T), human cervical cancer cells (HeLa), and HL-1 cells were purchased from Kebai Biotechnology Company in China. Mouse primary cardiomyocytes (MPCs) were isolated from cgas gene knockout (cgas- / -), Sting gene knockout (Sting- / -), cardiomyocyte-specific cgas gene knockout (cgas-CKO), cardiomyocyte-specific Sting gene knockout (Sting-CKO) mice, and their littermate control mice or C57BL / 6J wild-type (WT) mice. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS; 9014-81-7, Sigma-Aldrich, Germany). The cell culture environment temperature was maintained at 37 °C. Cell immunofluorescence staining and laser confocal analysis: During immunocytochemical staining, HeLa cells, HL-1 cells, or cardiomyocytes (CMs) were seeded on 14-mm coverslips (product of WHB Company, China) at a density of 3×10 5 cells per milliliter. After treatment with ligands, the cells were immediately fixed with 4% paraformaldehyde (product of Beyotime Institute of Biotechnology, China) for 5 minutes. Next, the cells were treated with 0.1% Triton X-100 (product of Beyotime Institute of Biotechnology, China) for 5 minutes to increase cell membrane permeability. Subsequently, the cells were incubated in a buffer containing 10% donkey serum (product of Solarbio Science & Technology Co., Ltd., China) at room temperature for 1 hour to block non-specific binding sites. Then, the samples were incubated with specific primary antibodies overnight at 4 °C. After that, the samples were rinsed three times with PBS (product of Solarbio Science & Technology Co., Ltd., China). The samples were then incubated with the designated fluorescent secondary antibodies at room temperature for 1 hour, followed by three more rinses with PBS. Finally, the samples were counterstained with 4',6-diamidino-2-phenylindole (DAPI, ab104139; Abcam, UK) to label the nuclei.

[0076] Statistical analysis: Data are presented as mean ± standard error of the mean (SEM). All statistical analyses were carefully performed using GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). The standard error is represented by error bars. First, the normal distribution of the data was examined, and subsequently, the Shapiro–Wilktest was applied to evaluate the homogeneity of variance. If the data showed an approximate normal distribution (P > 0.05), it indicated the applicability of parametric statistical methods. For normally distributed data, to evaluate the statistically significant differences between two groups, an unpaired two-tailed Student's t-test was used. For non-normally distributed data, non-parametric statistical methods were employed. Specifically, the Kruskal–Wallis test was used, followed by Dunn’s post-hoc test for multiple comparisons. The differences were judged to be statistically significant according to strict criteria: *P < 0.05 indicates marginal significance, **P < 0.01 indicates moderate significance, ***P < 0.001 indicates high significance, ****P < 0.0001 indicates extremely high significance, otherwise, it is considered no significant difference. At least three independent experiments were repeated for statistical analysis. Each data point included at least three biological replicates.

[0077] Results

[0078] 1. I / R induces the upregulation of cGAS-STING in cardiomyocytes

[0079] To initially determine the role of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-mediated DNA sensing in ischemia-reperfusion injury (IRI), we first detected the changes in cytoplasmic DNA levels. To clarify whether cardiac ischemia-reperfusion (I / R) causes cellular DNA damage, we initially established an ischemia-reperfusion model using 8-week-old male C57BL / 6J mice ( Figure 1 a-b), and analyzed the co-immunostaining and expression levels of double-stranded DNA (dsDNA) and cGAS in the ischemia-reperfusion border region. Compared with the sham-operated control group, a significant increase in cytoplasmic DNA content and activation of cGAS were observed in the cells of mice subjected to ischemia-reperfusion ( Figure 1c), indicating severe DNA damage in diseased tissues and DNA leakage into the cytoplasm. After recognizing double-stranded DNA, cGAS is activated. For further study, we used Western blotting to detect the levels of cGAS and stimulator of interferon genes (STING). The results showed that after 45 minutes of cardiac ischemia followed by 24 hours of reperfusion, the expressions of cGAS and STING were significantly upregulated in the ischemic-reperfusion border zone ( Figure 1 d-e). However, compared with the sham-operated group, there were no obvious changes in the protein levels of cGAS and STING in the infarct core region, and the levels of these two proteins were negligible in the non-ischemic region not affected by ischemia-reperfusion. Meanwhile, the results of immunofluorescence staining of tissue sections from the ischemia-reperfusion group and the sham-operated group showed that the expression of STING was significantly enhanced in the ischemic region (border zone) of the ischemia-reperfusion group, while there were no significant changes in the infarct core region or non-ischemic region.

[0080] In addition, to detect evidence of the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway after ischemia-reperfusion (I / R), we isolated cardiomyocytes (CMs), fibroblasts, and macrophage populations from the hearts of wild-type (WT), cgas knockout (cgas- / -), or Sting knockout (Sting- / -) mice according to the established protocol, which enabled us to study the specific roles of cGAS and STING in different cardiac cell types after ischemia-reperfusion. At the protein level, it was confirmed that the expressions of cGAS and STING were upregulated in cardiomyocytes in the border zone of the ischemia-reperfusion heart. Notably, in cardiomyocytes with cgas knockout or Sting knockout, these increased expressions were negligible ( Figure 1 f-g). However, no obvious differences were observed in macrophages or fibroblasts whether they had experienced ischemia-reperfusion or not ( Figure 1 f-g). Since the above results indicated that the role of cGAS in ischemia-reperfusion depends on STING, we then shifted our research focus to STING. Then, we designed an immunofluorescence co-localization experiment to further clarify the expression pattern of STING in cardiomyocytes. Immunofluorescence staining showed that compared with the negative control, the expression of STING was significantly upregulated in cardiomyocytes located in the border zone of the ischemia-reperfusion heart ( Figure 1 h).

[0081] To verify our research results in vitro, we established an oxygen-glucose deprivation / reoxygenation (A / R) model using mouse primary cardiomyocytes (MPCs) obtained from mouse hearts ( Figure 1i). Co-immunostaining analysis of live cell mitochondrial probes (mitochondrial tracers, MitoTracker) and double-stranded DNA (dsDNA) was performed to identify cytoplasmic DNA that does not co-localize with either the nucleus or mitochondria. Notably, compared with primary cardiomyocytes from control mice, we observed significant release of dsDNA after hypoxia / reoxygenation treatment, accompanied by severe mitochondrial damage, manifested as enlarged, shortened, and thickened mitochondria ( Figure 1 j). Collectively, these findings indicate that the double-stranded DNA (dsDNA)-cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway is activated in cardiomyocytes of the heart during ischemia-reperfusion (I / R). We shifted our research focus to understanding the specific roles of cGAS and STING in cardiomyocytes during ischemia-reperfusion injury (IRI).

[0082] 2. Knockout of cGAS-STING protects the heart from I / R injury

[0083] The roles of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) or stimulator of interferon genes (STING) in cardiomyocytes are not well understood. Next, we generated cardiomyocyte-specific cgas gene knockout mice [cgas fl / fl Myh6cre (cgas-CKO)] and Sting gene knockout mice [Sting fl / fl Myh6cre (Sting-CKO)] to investigate the roles and functions of cardiomyocyte-specific cGAS and STING in myocardial ischemia-reperfusion (MI / R) injury ( Figure 2 a). cgasfl / fl mice and cgasfl / fl Myh6cre mice, as well as Stingfl / fl mice and Stingfl / fl Myh6cre mice, were subjected to ischemia-reperfusion. Cardiac injury was evaluated by quantifying the necrotic area in heart sections, assessing cardiac function, and performing Masson staining. The cardiac function of knockout mice and their control mice at baseline, represented by left ventricular ejection fraction and fractional shortening (LVEF and FS, respectively), was comparable (P>0.05), and no significant differences were observed. These findings confirmed that there were no intrinsic abnormalities in the cardiac function of knockout mice before injury. According to the results, compared with control mice, cgas gene knockout (cgas-CKO) mice had a reduced necrotic area, significantly better cardiac function (ejection fraction and fractional shortening levels), and a decreased fibrotic area after ischemia-reperfusion ( Figure 2b-d), indicating that cardiomyocyte-specific cgas gene knockout has a protective effect on myocardial ischemia-reperfusion injury. Masson staining of heart tissues at 7 days of reperfusion showed reduced fibrosis in cgas gene knockout or Sting gene knockout (cgas- or Sting-CKO) mice.

[0084] To further clarify the role of cGAS in MI / R injury, we isolated cardiomyocytes (CMs) from cgas gene knockout (cgas-CKO) mice and their control mice in an in vivo warm ischemia-reperfusion model. Western blot analysis showed that the expression level of cGAS in cardiomyocytes was extremely low under physiological conditions, but its expression could be induced and upregulated after ischemia-reperfusion ( Figure 2 e). Importantly, the upregulation of cGAS in cardiomyocytes induced by ischemia-reperfusion did not occur in cardiomyocytes of cgas-CKO mice.

[0085] Interestingly, similar results were also observed in Sting gene knockout (Sting-CKO) mice ( Figure 2 f-h). As a downstream effector molecule of cyclic GMP-AMP synthase (cGAS), compared with control mice, the necrotic area was reduced, cardiac function was improved, and the fibrotic area was also decreased in Sting-CKO mice. Ischemia-reperfusion (I / R) was also able to induce the expression of stimulator of interferon genes (STING) in cardiomyocytes (CMs) of Stingfl / fl mice ( Figure 2 i). Notably, I / R had no effect on the STING protein level in cardiomyocytes of Sting-CKO mice. These findings indicate that the cGAS-STING signaling pathway plays a destructive role in cardiac injury after ischemia-reperfusion, and the exacerbating effect of cGAS on myocardial ischemia-reperfusion (MI / R) injury depends on the activation of STING.

[0086] 3. STING exacerbates ferroptosis of cardiomyocytes by regulating oxidative stress injury

[0087] To clarify the specific pathway by which stimulator of interferon genes (STING) causes myocardial ischemia-reperfusion (MI / R) injury, we performed RNA sequencing (RNA-seq) on heart sections of Sting gene knockout (Sting-CKO) mice and their control mice that underwent ischemia-reperfusion (IR) ( Figure 3 a). Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, it was found that among the differentially expressed genes (DEGs), genes involved in the cell growth and death pathway were significantly enriched ( Figure 3b). Therefore, we evaluated the degree of cell death in heart tissues and cardiomyocytes (CMs) derived from cgas gene knockout (cgas-CKO) or Sting-CKO mice and control mice subjected to ischemia-reperfusion (I / R) injury. Notably, the number of TUNEL-positive cells in the heart increased significantly after ischemia-reperfusion. In contrast, the levels of TUNEL-positive cells in cgas-CKO and Sting-CKO mice were significantly lower compared to their respective control groups ( Figure 3 c), indicating reduced cell death. Given that terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) can also detect DNA breaks caused by necrosis or other cell death processes, we used Western blotting to detect the expression levels of key cell death markers such as pro-caspase-3, cleaved-caspase-3, and B-cell lymphoma 2 (BCL-2). The results showed that the abundances of these proteins in Sting-CKO mice did not change significantly after ischemia-reperfusion compared to control mice. Collectively, these results confirm that the lack of STING alleviates cell death in the heart after ischemia-reperfusion injury, but not by regulating apoptosis.

[0088] Since cells have multiple ways of dying, according to Gene Ontology (GO) enrichment analysis, compared with Sting gene knockout (Sting-CKO) mice, the heart sections of Stingfl / fl mice showed significant changes in autophagolysosome, oxidative stress, and ferroptosis signaling pathways after ischemia-reperfusion (I / R). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining can also serve as a marker for ferroptosis. Next, we investigated the effect of stimulator of interferon genes (STING) on ferroptosis biomarkers, including lipid peroxidation, reactive oxygen species (ROS) accumulation, and the degradation of glutathione peroxidase 4 (GPX4), as well as the expression of some ferroptosis-related proteins. Recently, lipid peroxidation has been considered to play a direct role in promoting cell necrosis and ferroptosis. To explore whether STING regulates ferroptosis induced by hypoxia / reoxygenation (A / R), we detected the production of malondialdehyde (MDA), which is the end product of lipid peroxidation. The results showed that STING could very effectively promote the production of MDA after hypoxia / reoxygenation treatment, with an effect comparable to that of the positive control drug Erastin ( Figure 3e), Meanwhile, the activation of STING blocked the inhibitory effect of ferroptosis inhibitor-1 (Fer-1) on MDA production in ferroptosis. These observations indicate that STING acts upstream of the lipid peroxidation process and thus plays a key role in the regulation of ferroptosis.

[0089] Given that mitochondrial damage and oxidative stress response are key events during ischemia-reperfusion (I / R), to further verify the relationship between stimulator of interferon genes (STING) and cardiac oxidative stress, we first confirmed the presence of oxidative stress injury by detecting the production of reactive oxygen species (ROS) in cardiomyocytes (CMs) after hypoxia / reoxygenation (A / R). Exposure of cultured cardiomyocytes to hypoxia / reoxygenation triggers an increase in ROS production. Before live cell imaging, primary mouse cardiomyocytes (MPCs) were pretreated with ferroptosis inhibitor-1 (Fer-1) for 12 hours to reduce ROS levels, and then cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) was added at 30 minutes and 36 seconds to activate STING. Observation and imaging were stopped once the ROS level stabilized ( Figure 3 f). Interestingly, in the presence of cGAMP, the inhibitory effect of Fer-1 on ROS in ferroptosis was significantly disrupted. The results indicate that the activation of STING leads to an increase in ROS production.

[0090] After analyzing the effects of STING on ferroptosis biomarkers including lipid peroxidation and ROS accumulation, we next aimed to determine the potential mechanism by which STING promotes ferroptosis by evaluating the abundance of lipid peroxidation- and ferroptosis-related factors. We used Western blotting to evaluate the expression abundance of acyl-CoA synthetase long-chain family member 4 (ACSL4), transferrin receptor (TFR), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4), which have all been identified as protein targets of ferroptosis. We observed that compared with the control group, the expression of ACSL4 and TFR decreased, while the expression of GPX4 increased and SLC7A11 showed no significant change in Sting gene knockout (Sting-CKO) mice after ischemia-reperfusion ( Figure 3 g-h). The upregulation of ferroptosis was further confirmed at the protein level in cardiac progenitor cells (MPCs) after hypoxia-reoxygenation (A / R) treatment. Figure 3(i-j). Interestingly, during A / R-triggered ferroptosis, the absence of Sting blocked the degradation of GPX4, and the degradation of GPX4, as a key inhibitor of lipid peroxidation, is crucial for promoting lipid peroxidation in ferroptosis. Next, we measured the levels of GPX4 messenger ribonucleic acid (mRNA) in the heart tissues of sham-operated and I / R-treated mice using real-time fluorescence quantitative polymerase chain reaction (qPCR). Our results showed that there was no change in the GPX4 mRNA levels between the Sting wild-type (Stingfl / fl) and Sting-CKO groups after I / R treatment, indicating that the decrease in GPX4 protein was not due to transcriptional downregulation but due to post-translational regulation. In summary, we concluded that STING is a potent activator of ferroptosis, which can promote GPX4 protein degradation, lipid peroxidation, and ROS accumulation. Subsequently, we delved deeper into the molecular events associated with STING and the reduction of GPX4 mediated by I / R.

[0091] 4. STING targets GPX4

[0092] The upregulation of the ferroptosis program after the activation of stimulator of interferon genes (STING) implies that under normal circumstances, myocardial ischemia-reperfusion (MI / R) injury may promote the accidental activation of the signal transduction cascade involving STING. We wondered what genetic factors might be involved and whether we could utilize these factors to intervene in the exacerbation of myocardial ischemia-reperfusion injury in STING-activated mice. First, we analyzed the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways expressed in the differentially expressed genes (DEGs) identified by RNA sequencing ( Figure 3 (a). Notably, among these differentially expressed genes, ferroptosis-related genes were significantly enriched ( Figure 4 (a). Next, in mouse primary cardiomyocytes (MPCs), tandem affinity purification was performed using a STING protein affinity antibody to determine the molecular mechanism by which STING regulates ferroptosis. Silver staining was performed on the immunopurified material ( Figure 4 (b i), followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS / MS) ( Figure 4 (b ii) to identify STING protein partners. This approach revealed several proteins, especially the ferroptosis-related factor glutathione peroxidase 4 (GPX4). The predicted results of molecular docking analysis between STING (Protein Data Bank [PDB] ID: 4F5W) and GPX4 (PDB ID: 5L71) were shown using PyMOL software. Figure 4 (c) depicts ten potential binding modes with a matching alignment score of 8.157, which increases the likelihood that STING may directly interact with GPX4.

[0093] To further confirm the direct interaction between stimulator of interferon genes (STING) and glutathione peroxidase 4 (GPX4), we used a STING-specific antibody to precipitate its protein partners, and then combined this protein purification product with GPX4 antibody and monitored it in immunoprecipitation experiments. Endogenous co-immunoprecipitation experiments showed that endogenous STING and GPX4 formed a complex in cardiomyocytes (CMs) after hypoxia-reoxygenation (A / R), and cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) further promoted the formation of this complex ( Figure 4 d). Correspondingly, STING could be detected in the endogenous purification product of GPX4 ( Figure 4 e), indicating a direct interaction between these two proteins. Next, we expressed Myc-tagged STING (Myc-STING) and Flag-tagged GPX4 (Flag-GPX4) plasmids in HeLa cells stably overexpressing these constructs. Then Myc or Flag immunoprecipitation experiments were performed to study the interaction between Myc-STING and Flag-GPX4. The input samples and eluates were analyzed by Western blotting, and the results showed that enrichment of Myc-STING could be detected in the purification product eluted with the Flag peptide of excess Flag-GPX4 ( Figure 4 f). Correspondingly, enrichment of Flag-GPX4 was also observed in the purification product of Myc-STING ( Figure 4 g). In addition, we found that the interaction between GPX4 and STING was enhanced in a time-dependent manner in cGAMP-treated HeLa cells ( Figure 4 h), while erastin weakened the intensity of this interaction.

[0094] To confirm the interaction between stimulator of interferon genes (STING) and glutathione peroxidase 4 (GPX4), we performed immunostaining experiments to analyze the co-localization of STING and GPX4 in mouse primary cardiomyocytes (MPCs). Under normoxic conditions, the expression level of STING in mouse primary cardiomyocytes was low and there was no co-localization with GPX4 ( Figure 4 i). After acute hypoxia-reoxygenation (A / R), STING was activated and formed aggregates with GPX4 ( Figure 4 i), and upon stimulation with cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), the two aggregated in large numbers and showed a co-localized state ( Figure 4 i). Interestingly, after treatment with erastin, there was almost no co-localization between GPX4 and STING (Figure 4 i). These findings are highly consistent with the experimental results obtained from co-immunoprecipitation (CoIP) studies. Taken together, these evidences strongly suggest that STING and GPX4 directly interact in cardiomyocytes after hypoxia-reoxygenation. Additionally, as the activation degree of STING by cGAMP increases, a more obvious clustered distribution of STING and GPX4 can be observed, indicating an enhanced binding between these two proteins.

[0095] Given the known co-localization and interaction between stimulator of interferon genes (STING) and glutathione peroxidase 4 (GPX4) in mouse and human cells, combined with the previously mentioned role of STING in regulating ferroptosis, we hypothesized that GPX4 might be involved in the regulation of ischemia-reperfusion (I / R) injury caused by STING. Immunostaining analysis of heart sections from Stingfl / fl mice and Sting knockout (Sting-CKO) mice revealed that in the ischemia-reperfusion junction region of the Stingfl / fl mouse heart, ischemia-reperfusion (I / R) induced enhanced STING expression and co-localization of STING and GPX4 ( Figure 4 j). Conversely, when Sting was specifically knocked out, the signal of GPX4 became much brighter and there was no co-localization with STING. These results further confirmed the previously discovered role of STING in inducing ferroptosis and the binding between STING and GPX4 after ischemia-reperfusion (IR). It is worth noting that this binding may lead to downregulation of GPX4 expression, which requires further research to explore in depth.

[0096] In summary, there is a direct interaction between stimulator of interferon genes (STING) and glutathione peroxidase 4 (GPX4), and the STING activator cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) coordinates the interaction between GPX4 and STING. This effect exists in both mouse and human cell lines. Since the deletion of the Sting gene actually led to an increase in GPX4 expression in heart sections after ischemia-reperfusion (IR), which is consistent with previous studies showing that STING can induce ferroptosis. Based on these observations, we speculate that STING may directly bind to GPX4, thereby inhibiting its activity and ultimately leading to the occurrence of ferroptosis.

[0097] 5. STING and GPX4 directly interact at the 146th amino acid residue (N146) of GPX4 and the 267th amino acid residue (T267) of STING

[0098] To further clarify the binding sites between stimulator of interferon genes (STING) and glutathione peroxidase 4 (GPX4), we conducted a detailed study on the images and related data in the Protein Data Bank (PDB). We selected the ligand-free STING (Apo STING) oligomer (PDB ID: 4F5W) and GPX4 (PDB ID: 5L71) for protein interaction prediction. The prediction results showed that STING might dock with GPX4 at amino acid sites such as Y167, E260, Y245, Q266, or T267. Correspondingly, GPX4 might dock with STING at amino acid sites such as G126, R127, or N146. Figure 5 a). Based on the above predictions, we performed immunoprecipitation experiments on HeLa cells expressing Flag-tagged GPX4 (Flag-GPX4) mutants and Myc-tagged STING (Myc-STING) mutants to determine the interaction domains between STING and GPX4. First, we evaluated three GPX4 peptides, namely ΔG126, ΔR127, and ΔN146. By analyzing the results of protein immunoblotting, we found that only the Flag-GPX4-ΔN146 mutant lost the ability to interact with STING, indicating that this amino acid plays a key role in the interaction between these two proteins. Figure 5 b). On the other hand, we constructed Myc-tagged STING mutants lacking specific amino acids, namely Myc-STING-ΔY167, Myc-STING-ΔE260, Myc-STING-ΔY245, Myc-STING-ΔQ266, and Myc-STING-ΔT267. The results showed that the deletion of the T267 amino acid in STING eliminated the interaction between STING and GPX4. Figure 5 c).

[0099] In addition, in HeLa cells stably overexpressing Myc-tagged stimulator of interferon genes (STING) and Flag-tagged glutathione peroxidase 4 (GPX4), we observed that when the T267 site of STING was deleted, even though the whole genome of GPX4 was overexpressed, GPX4 lost the co-localization with STING. Figure 5 d). Similarly, when GPX4 lacked the N146 site, the interaction between STING and GPX4 also disappeared. Figure 5 d). Taken together, these data conclusively demonstrated that STING physically interacts with the N146 site of GPX4 through its own T267 amino acid site.

[0100] To enhance the clarity of immunostaining images using a super-resolution imaging microscope (SIM) device, we observed a strong binding and co-localization between stimulator of interferon genes (STING) and glutathione peroxidase 4 (GPX4) in HL-1 cells transfected with Myc-tagged STING and Flag-tagged GPX4 plasmids. By this method, we were able to more precisely understand the interaction pattern between these two proteins in the cellular environment ( Figure 5 e). Delivery of cGAMP further accelerated the formation of the STING-GPX4 complex. Notably, when Flag-GPX4-ΔN146 and Myc-STING were co-transfected simultaneously, GPX4 lost its co-localization with STING. At this time, even when cGAMP was delivered, the co-localization between these two proteins could not be restored, highlighting the crucial role of this amino acid in promoting the interaction between these two proteins.

[0101] 6. Stimulator of interferon genes (STING) promotes ferroptosis through autophagolysosome-mediated degradation of glutathione peroxidase 4 (GPX4)

[0102] Degradation of glutathione peroxidase 4 (GPX4) protein is a key event in ferroptosis, which in turn promotes the production of reactive oxygen species (ROS) and leads to irreversible lipid peroxidation, ultimately resulting in cell death. In Figure 4 j, we also observed the potential degradation of GPX4 by stimulator of interferon genes (STING) in cardiac tissue. Next, we verified the effect of STING on GPX4 degradation during ferroptosis. Given that we observed that STING deficiency blocked the degradation of GPX4 during ferroptosis induced by ischemia / reperfusion (I / R) or anoxia / reoxygenation (A / R), we further explored the stimulatory effect of the STING activator cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) on GPX4 degradation. Notably, we observed that, in contrast to the negative control ferroptosis inhibitor-1 (Fer-1), cGAMP led to a decrease in the GPX4 protein level ( Figure 6 a). Treatment of myocardial progenitor cells (MPCs) with the ferroptosis inhibitor Fer-1 led to an increase in the GPX4 protein level, but this increase was blocked in the presence of cGAMP. In addition, Erastin, which can inhibit the activity of GPX4, had a similar effect to the aforementioned cGAMP and led to the accumulation of intracellular lipid peroxides. To evaluate whether cGAMP enhanced the degradation of GPX4, we used Erastin in the experiment. The use of cGAMP did trigger the degradation of GPX4 ( Figure 6b). In addition, the addition of H-151 can also protect cells from ferroptosis and inhibit the degradation of GPX4 induced by Erastin. In summary, the activation of STING can trigger the degradation of GPX4.

[0103] Subsequently, we deeply explored the potential mechanism by which stimulator of interferon genes (STING) regulates the degradation of glutathione peroxidase 4 (GPX4). The degradation of post-transcriptional proteins mainly occurs through two mechanisms: ubiquitin-mediated proteasomal degradation and autophagy-mediated lysosomal degradation. Figure 3 The research results in d also showed that during ischemia-reperfusion (I / R) injury, the autophagy-lysosome pathway was enriched in STINGfl / fl mice, indicating that STING may play a regulatory role in the degradation of GPX4 through the autophagy pathway. Therefore, we used several protein degradation inhibitors, including proteasome inhibitors (MG-132 and calpain inhibitor) and lysosomal inhibitors (chloroquine (CQ) and ammonium chloride (NH4Cl)) to specifically block these degradation pathways. Interestingly, only the lysosomal inhibitors CQ or NH4Cl could effectively block the degradation of GPX4 induced by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and increase the content of GPX4 ( Figure 6 c), indicating that they are involved in the regulation of GPX4 homeostasis during hypoxia-reoxygenation (A / R) exposure. Bafilomycin A1 (Baf A-1) is an inhibitor of the late stage of autophagy, which effectively prevents the maturation of autophagosomes by disrupting the fusion process between autophagosomes and lysosomes. LY294002, 3-methyladenine (3-MA) and wortmannin are inhibitors targeting the initiation stage of autophagy and can effectively block the formation of autophagosomes. Through Western blot analysis, we found that only Baf A-1 could block the degradation of GPX4 induced by cGAMP ( Figure 6 d), indicating that STING promotes the autophagic degradation of GPX4 by promoting the fusion of autophagosomes and lysosomes.

[0104] Autophagy is a key cellular process responsible for protein degradation. It has become an important potential factor mediating the reduction of glutathione peroxidase 4 (GPX4) induced by stimulator of interferon genes (STING) after ischemia-reperfusion (I / R). The deletion of STING can hinder autophagy triggered by I / R. To further confirm that STING intervenes in the autophagic degradation of GPX4 by regulating the binding of autophagosomes and lysosomes, we used the RFP-GFP-LC3 double-fluorescence autophagy indicator system to carefully label and track the changes in LC3 and autophagic flux. Preliminary observation results showed that autophagy was strongly activated under hypoxia-reoxygenation (A / R) conditions. Figure 6e-g). The chimeric fluorescent protein-LC3 is firmly attached to the autophagosomal membrane and fuses with lysosomes to form autophagolysosomes. Notably, during this process, the fluorescence of green fluorescent protein (GFP) weakens, indicating that autophagosomes have successfully transformed into autophagolysosomes. However, in Sting gene knockout (Sting-CKO) myocardial progenitor cells (MPCs), a significant increase in yellow fluorescent dots was observed, while there were fewer red fluorescent dots. This phenomenon strongly confirmed the results obtained from our Western blot experiments, indicating that the fusion of autophagosomes with lysosomes was hindered and the maturation process of autophagosomes was disrupted. Essentially, the deletion of Sting prevented the clearance of GPX4 by blocking the binding of autophagosomes to lysosomes.

[0105] Stimulator of interferon genes (STING) is well-known for its ability to initiate autophagy through the lipidation of microtubule-associated protein 1A / 1B light chain 3B (LC3B) and subsequently form autophagosomes, and this process is independent of the activation of TANK-binding kinase 1 (TBK1) and the induction of interferons. Next, we investigated the roles of STING, LC3B, and glutathione peroxidase 4 (GPX4) in ferroptosis. Using triple immunofluorescence analysis, we observed that under hypoxia-reoxygenation (A / R) conditions, STING was activated, leading to the formation of LC3 puncta in myocardial progenitor cells (MPCs) of Stingfl / fl mice, but not in Sting-deficient cells ( Figure 6 h). This finding was consistent with the results reported in previous literature. In Stingfl / fl MPCs, significant co-localization of LC3 puncta with both STING and GPX4 was observed, but not in cells lacking STING, indicating that GPX4 is the target of STING-induced autophagy after A / R. To determine the location of the interaction between STING and GPX4, we co-stained STING, GPX4, and markers of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), coat protein I (COP-I), and LC3 in MPCs. Our results showed that the STING-GPX4 complex was initially localized to the ERGIC. Subsequently, the STING-GPX4 complex was transported to the ERGIC, COP-I vesicles, and LC3 autophagosomes.

[0106] In addition, lysosome-associated membrane protein 2B (LAMP2B) mediates the mechanism of autophagosome-lysosome fusion. Autophagosomes formed by hypoxia-reoxygenation (A / R) stimulation can be observed through LAMP2B. The imaging results showed that the GPX4-STING puncta induced by A / R were surrounded by the autophagolysosomal membrane ring labeled by LAMP2B ( Figure 6i). This indicates that the interaction between STING and GPX4 occurs in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and STING promotes the recruitment of GPX4 to autophagosomes for subsequent autophagic degradation. These findings suggest that STING-induced autophagy contributes to the clearance of GPX4 in cardiomyocytes (CMs) after A / R.

[0107] 7. Adeno-associated virus (AAV)-mediated glutathione peroxidase 4 (GPX4) therapy protects cardiac function from severe ischemia-reperfusion (I / R) injury triggered by stimulator of interferon genes (STING) activation

[0108] Based on our research findings, we subsequently confirmed that glutathione peroxidase 4 (GPX4) is a downstream factor of stimulator of interferon genes (STING) and further explored the potential of GPX4 as a therapeutic target for STING-related ischemia-reperfusion (I / R) injury. We constructed an adeno-associated virus targeting GPX4 (AAV-GPX4, designated cTNT-GPX4) driven by the troponin T (cTNT) promoter in cardiomyocytes and labeled with green fluorescent protein (GFP) to investigate whether overexpression of GPX4 could alleviate the progressive decline of cardiac function in STING-activated mice under in vivo conditions. In STING-activated mice and their corresponding control mice, AAV-GPX4-GFP was injected via the tail vein 14 days before inducing sham ischemia-reperfusion (I / R). Subsequently, 5,6-dimethylxanthine-4-oxide (DMXAA, a STING activator) or dimethyl sulfoxide (DMSO) was intraperitoneally injected daily for three consecutive days before the I / R surgery ( Figure 7 a). Before establishing the model, the overexpression efficiency of GPX4 in cardiomyocytes isolated from three or six mice was confirmed by Western blotting and immunofluorescence staining ( Figure 7 b). Administration of the STING activator DMXAA increased the infarct area, while overexpression of GPX4 minimized the infarct area in STING-activated mice ( Figure 7 c-d), indicating that GPX4 overexpression has a protective effect on STING activation-induced I / R injury. Echocardiographic analysis showed that STING activation did exacerbate cardiac dysfunction after I / R. However, in STING-activated mice administered DMXAA, overexpression of GPX4 significantly improved the deteriorating cardiac function, as evidenced by the improvement in the reduction of ejection fraction (EF) and fractional shortening (FS) after ischemia-reperfusion (I / R) ( Figure 7 e-f).

[0109] 4-Hydroxy-2-nonenal (4-HNE) is a widely recognized byproduct of lipid peroxidation and a key indicator of ferroptosis. Our research results show that the activation of stimulator of interferon genes (STING) leads to a significant increase in 4-HNE levels, indicating enhanced lipid peroxidation( Figure 7 g), while the overexpression of glutathione peroxidase 4 (GPX4) can reverse the increased lipid peroxidation caused by 5,6-dimethylxanthine-4-oxide (DMXAA), suggesting a downregulation of ferroptosis. In vitro detection of the reactive oxygen species (ROS) accumulation level in myocardial progenitor cells (MPCs) strongly confirmed this observation( Figure 7 h). To further explore the effect of the cyclic GMP-AMP synthase (cGAS)-STING-GPX4 axis on mitochondria, we conducted a series of experiments. These experiments included measuring adenosine triphosphate (ATP) levels, evaluating mitochondrial membrane potential (ΔΨm), and determining oxygen consumption rate (OCR) to assess the health status, ATP content, membrane potential, and respiratory capacity of mitochondria under hypoxia-reoxygenation (A / R) conditions. The results showed that blocking the cGAS / STING signaling pathway or overexpressing GPX4 could partially restore ATP levels, mitochondrial membrane potential (ΔΨm), basal mitochondrial respiration, ATP production, as well as maximal and spare respiratory capacities, indicating that mitochondrial function could be partially restored by knocking out cGAS / STING or overexpressing GPX4.

[0110] In summary, it can be concluded that although ischemia-reperfusion (I / R) injury is exacerbated in STING-overexpressing mice, the upregulation of glutathione peroxidase 4 (GPX4) still has a significant ability to inhibit lipid peroxidation and ferroptosis, thereby protecting cardiac function. In general, knocking out cyclic GMP-AMP synthase (cGAS) / stimulator of interferon genes (STING) or overexpressing GPX4 can protect STING-activated mice from the deterioration of cardiac dysfunction and mitochondrial damage after I / R, which points out a potential therapeutic strategy for alleviating the adverse effects of STING overexpression on cardiac function.

[0111] 8. Stimulator of interferon genes (STING) is a potential therapeutic target for inhibiting cardiac dysfunction after ischemia-reperfusion (I / R)

[0112] To explore more deeply the ischemic reperfusion (I / R) treatment strategy utilizing the stimulator of interferon genes (STING)-glutathione peroxidase 4 (GPX4) signaling pathway, we selected an STING inhibitor, H-151, to evaluate its potential in alleviating the harmful effects of I / R on cardiac function and to search for potential therapeutic drugs for treating I / R injury. Before the I / R surgery, mice were intraperitoneally injected with H-151 (5 mg / kg.bw) every two days for 7 days ( Figure 8 a). Meanwhile, the degree of myocardial necrosis was clearly visible in in vivo cardiac images ( Figure 8 a), indicating that H-151 could effectively control the necrotic area within the cardiac function region. Notably, with the use of H-151, a reduced infarct area was observed ( Figure 8 b-c) and cardiac function was restored ( Figure 8 d-e). In addition, the addition of H-151 could alleviate the elevated level of 4-hydroxy-2-nonenal (4-HNE) induced by I / R, indicating a reduced degree of lipid peroxidation and ferroptosis ( Figure 8 f).

[0113] Subsequently, we further explored the pharmacological mechanism by which H-151 improved myocardial injury after I / R in vitro. The results showed that H-151 blocked the interaction between stimulator of interferon genes (STING) and glutathione peroxidase 4 (GPX4) ( Figure 8 g), blocked autophagic flux ( Figure 8 h), thereby reducing the accumulation of reactive oxygen species (ROS) ( Figure 8 i), and further alleviating ferroptosis of cardiomyocytes.

[0114] Meanwhile, to further enhance the therapeutic effect, we evaluated the efficacy of combining the STING inhibitor H-151 with the autophagy inhibitor bafilomycin A1. Before the I / R surgery, mice were intraperitoneally injected with H–151 (5 mg / kg.bw) and bafilomycin A1 (2 mg / kg.bw) simultaneously every two days for 7 days; it was found that the combination of the two could more effectively reduce the necrotic area after myocardial ischemia reperfusion ( Figure 8 j), the cardiac function (ejection fraction and fractional shortening levels) recovered significantly better ( Figure 8 k), the fibrotic area was significantly reduced, and the elevated level of 4-hydroxy-2-nonenal (4-HNE) induced by I / R was more effectively alleviated ( Figure 8 l), and the effect was significantly better than that of each of them used alone, thus producing a good synergistic effect against myocardial ischemia reperfusion injury.

[0115] In summary, stimulator of interferon genes (STING) has emerged as a promising therapeutic target for alleviating cardiac injury that occurs after ischemia-reperfusion (IR). By regulating glutathione peroxidase 4 (GPX4), the harmful effects brought about by STING can be controlled, which provides a promising avenue for intervening in cardiac remodeling.

[0116] Matters not covered in this invention are well-known techniques.

[0117] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression levels of STING and its related biomolecules in the preparation of a product for detecting myocardial ischemia-reperfusion injury; wherein, The related biomolecules of STING include any one or more of double-stranded DNA, cGAS, cGAMP, and GPX4.

2. The application according to claim 1, characterized in that, The myocardial ischemia-reperfusion injury detection product is used for the screening, (auxiliary) diagnosis, monitoring, or prognosis evaluation of myocardial ischemia-reperfusion injury.

3. The application according to claim 1, characterized in that, The substances for detecting the expression levels of STING and its related biomolecules include reagents for detecting the expression levels of the coding genes of STING and its related biomolecules based on real-time fluorescence quantitative PCR, in situ hybridization, gene chips, and gene sequencing, and / or reagents for detecting the protein (enzyme) expression levels of STING and its related biomolecules based on immunoassay methods; The product is a primer, probe, (gene or protein) chip, detection kit, and detection device.

4. A system for detecting myocardial ischemia-reperfusion injury, characterized in that, The system includes: An acquisition unit configured to acquire the expression level conditions of STING and its related biomolecules of a subject; An analysis unit configured to analyze and judge the disease condition of the subject according to the expression level conditions of STING and its related biomolecules of the subject obtained by the acquisition unit; Among them, the related biomolecules of STING include any one or more of double-stranded DNA, cGAS, cGAMP, and GPX4.

5. The system according to claim 4, characterized in that, The sample to be tested of the subject is a myocardial cell sample of the subject; The detection of myocardial ischemia-reperfusion injury is for the screening, (auxiliary) diagnosis, monitoring, or prognosis of myocardial ischemia-reperfusion injury.

6. Use of STING and its related biomolecules as targets in the preparation and / or screening of drugs for myocardial ischemia-reperfusion injury; wherein, The related biomolecules of STING include GPX4.

7. The application according to claim 6, wherein The method for screening drugs for preventing and / or treating myocardial ischemia-reperfusion injury includes: (I) Treating a system expressing and / or containing STING and its related biomolecules with a candidate substance; setting a control without treating with the candidate substance; (II) After completing step (I), detecting the expression levels of STING and its related biomolecules in the system; compared with the control, if the expression levels of STING and its related biomolecules in the system treated with the candidate substance change significantly, the candidate substance can be used as a candidate drug for preventing or treating myocardial ischemia-reperfusion injury; Furthermore, the specific method of step (II) is: if the expression level of STING in the system treated with the candidate substance is significantly down-regulated and / or the expression level of GPX4 is significantly up-regulated, the candidate substance is used as a candidate drug for preventing and / or treating myocardial ischemia-reperfusion injury.

8. Use of substances that inhibit the expression of STING or reduce its activity in any one or more of the following: (a) Inhibiting the degradation of GPX4 or preparing a product for inhibiting the degradation of GPX4; (b) Inhibiting ferroptosis or preparing a product for inhibiting ferroptosis; (c) A product for preventing and / or treating myocardial ischemia-reperfusion injury; Among them, Substances that inhibit the expression of STING or reduce its activity include RNA interference molecules or antisense oligonucleotides against STING, small molecule inhibitors (including H-151), siRNA, shRNA, substances for implementing lentiviral infection or gene knockout; Among them, in (a)-(c), the product acts on myocardial cells, myocardial tissues, and / or the heart.

9. The application according to claim 8, characterized in that The product is a drug or an experimental reagent for non-medical use.

10. Use of a substance that inhibits STING expression or reduces its activity in combination with a substance that inhibits the degradation of GPX4 in the preparation of a drug for preventing and / or treating myocardial ischemia-reperfusion injury; Among them, The substance that inhibits STING expression or reduces its activity is H-151, and the substance that inhibits the degradation of GPX4 is bafilomycin A1.

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