Use of rig-i inhibitors in myocardial ischemia reperfusion injury

Abstract

The application discloses application of a RIG-I inhibitor in myocardial ischemia-reperfusion injury. By conditionally knocking out a RIG-I gene in myocardial cells, it is found that RIG-I protein in myocardial cells participates in and relieves myocardial ischemia-reperfusion injury of mice, and it is determined that retinoic acid-inducible gene I (RIG-I, also known as DDX58) is a key treatment target. Further, by computer-aided drug design, a RIG-I specific small molecule inhibitor RI-Q1 is screened and obtained, and it is proved that the small molecule inhibitor can effectively inhibit pyroptosis inflammation of myocardial cells, thereby preventing or treating MI / RI. The small molecule inhibitor RI-Q1 is easy to obtain, can directly inhibit RIG-I protein activity, and significantly reduces pyroptosis and inflammatory reaction of myocardial cells. Therefore, as a novel small molecule compound targeting RIG-I, the small molecule inhibitor RI-Q1 provides a new drug development direction and application prospect for treatment of MI / RI.

Description

Technical Field

[0001] This invention relates to the application of RIG-I inhibitors in myocardial ischemia-reperfusion injury, and belongs to the field of biomedical technology. Background Technology

[0002] Currently, percutaneous coronary intervention (PCI) to restore myocardial blood flow reperfusion is the most widely recognized and effective treatment for reducing infarct size and mortality. However, reperfusion of blood flow into the patient's blood vessels can lead to further expansion of the infarct area and cardiomyocyte death, a condition known as myocardial ischemia / reperfusion injury (MI / RI). While various approaches and strategies for preventing and treating MI / RI have been proposed by scholars worldwide, it remains difficult to avoid and often induces complications such as arrhythmias, myocardial stifling, no-reflow, and microcirculatory disturbances, ultimately leading to adverse outcomes. Recent studies have found that RIG-I-mediated inflammation plays a crucial regulatory role in the development and complications of heart failure and atherosclerosis in humans. However, there are no reports on RIG-I as a therapeutic target for MI / RI, or on the development of corresponding small molecule compounds based on this target. Summary of the Invention

[0003] The purpose of this invention is to provide the application of RIG-I inhibitors and their small molecule compound RI-Q1 in myocardial ischemia-reperfusion injury. This invention demonstrates that conditional gene knockout of RIG-I in cardiomyocytes can effectively improve myocardial ischemia-reperfusion injury. Furthermore, using the virtual screening method in computer-aided drug design, the RIG-I-specific small molecule inhibitor RI-Q1 was obtained through high-throughput screening. RI-Q1 treats myocardial ischemia-reperfusion injury by inhibiting pyroptotic inflammation, thus developing the pharmaceutical applications of RI-Q1.

[0004] To achieve the above objectives, in a first aspect, the present invention provides the use of RIG-I as a detection target in screening drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0005] Secondly, the present invention provides the use of RIG-I inhibitors in the preparation of medicaments for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

[0006] In some embodiments, the RIG-I inhibitor includes: i) any one or more of shRNA, siRNA, ASO, and delivery vectors of said shRNA, siRNA, ASO, and reagents of the CRISPR / Cas system and Cre-loxP system that specifically inhibit RIG-I expression in myocardial tissue or cardiomyocytes; ii) any one or more of neutralizing antibodies, aptamers, and small molecule inhibitors that specifically bind to RIG-I.

[0007] In some embodiments, the delivery vector includes a viral vector or a non-viral vector.

[0008] In some embodiments, the viral vector is selected from one or more combinations of retroviral vectors, adenovirus vectors, and adeno-associated virus vectors; and / or, the non-viral vector includes mRNA liposomes.

[0009] In some embodiments, the RIG-I inhibitor is a reagent (including Cre recombinase) required for conditional knockout of RIG-I in cardiomyocytes via the Cre-loxP system.

[0010] In some embodiments, the RIG-I inhibitor is a small molecule compound RI-Q1 with the molecular formula: C 12 H 10 ClN3O3S has the following chemical structural formula:

[0011]

[0012] In some embodiments, the drug comprises an active ingredient and pharmaceutically acceptable excipients, the active ingredient including a RIG-I inhibitor.

[0013] In some embodiments, the pharmaceutically acceptable excipient is one or more of the following: diluent, binder, wetting agent, lubricant, disintegrant, solvent, emulsifier, cosolvent, preservative, pH adjuster, osmotic pressure adjuster, surfactant, coating material, antioxidant, and buffer. In some embodiments, the dosage form of the drug includes any one of injections, capsules (including sustained-release or delayed-release forms), tablets, pills, suspensions, granules, tinctures, syrups, and emulsions.

[0014] Thirdly, the present invention provides a RIG-I inhibitor with anti-pyroptosis activity, wherein the RIG-I inhibitor is formulated from RI-Q1 and pharmaceutically acceptable excipients.

[0015] The pharmaceutically acceptable excipients include, but are not limited to, pharmaceutically acceptable carriers, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, or 0.9% sodium chloride. "Excipients" refers to conventional pharmaceutical carriers, such as diluents, excipients like water, fillers like starch and sucrose, binders like cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone, humectants like glycerin, disintegrants like agar, calcium carbonate, and sodium bicarbonate, absorption enhancers like quaternary ammonium compounds, surfactants like hexadecyl alcohol, adsorbents like kaolin and soap clay, and lubricants like talc, calcium / magnesium stearate, and polyethylene glycol.

[0016] RI-Q1 is a RIG-I specific small molecule inhibitor obtained through high-throughput screening using virtual screening methods in computer-aided drug design. It exhibits excellent activity in inhibiting pyroptotic inflammatory responses and resisting myocardial ischemia-reperfusion injury. It can directly bind to RIG-I proteins, inhibiting their molecular interaction with Caspase-1 proteins, thereby suppressing the release of pyroptosis effectors and downstream inflammatory factors. Currently, there is a lack of drugs in clinical practice that treat myocardial ischemia-reperfusion injury by inhibiting pyroptosis. This invention, on the one hand, reveals the target and mechanism of RI-Q1 in inhibiting pyroptosis, and on the other hand, provides new drugs and theoretical basis for the treatment of myocardial ischemia-reperfusion injury.

[0017] Compared with the prior art, this application has the following beneficial effects: The RI-Q1 screened in this invention, as a specific small molecule inhibitor of RIG-I, can specifically target RIG-I, namely retinoic acid-induced protein 1, thereby inhibiting pyroptosis. It has excellent anti-inflammatory activity and can prevent or treat myocardial ischemia-reperfusion injury. As a drug for treating myocardial ischemia-reperfusion injury, it has significant effects. Attached Figure Description

[0018] Figure 1 The results show the effect of conditional RIG-I knockout of cardiomyocytes on the degree of acute myocardial ischemia-reperfusion injury in mice. Among them, A: schematic diagram of animal experimental process; BD: Evans blue and TTC double staining results (B) and corresponding quantitative statistical results of the ratio of myocardial ischemia area to left ventricle (AAR / LV, C) and the percentage of infarct area to ischemic area (INF / AAV, D); EF: TUNEL staining results of mouse ischemia-reperfusion heart tissue (E) and corresponding quantitative statistical results of apoptotic cell ratio (F); GI: mouse serum supernatant LDH (G), cTnT (H), CK-MB (I) content.

[0019] Figure 2The results show the effects of the small molecule compound RI-Q1 described in this invention on the hypoxia-reoxygenation model of mouse HL-1 cell line; wherein, A: schematic diagram of the construction of the cell hypoxia-reoxygenation model (H / R); B: result of cell viability detection after H / R modeling by CCK-8 assay; D: result of LDH content in the supernatant released by cells by LDH detection; E: morphological changes of cells in each group after H / R modeling observed under a 10× light microscope.

[0020] Figure 3 The diagram shows the structure and related molecular mechanism of the binding of the small molecule compound RI-Q1 to the RIG-I protein described in this invention. Among them, A: Surface plasmon resonance (SPR) experiment shows the fitting analysis of the binding of RI-Q1 and RIG-I; BE: Pull-Down experiments were used to verify the binding of RIG-I protein and RI-Q1 in purified RIG-I protein (B), HL-1 cell line (C), ischemic myocardial tissue after PCI in patients with ischemic cardiomyopathy (D), and ischemic-reperfusion area cardiac tissue in mice (E).

[0021] Figure 4 The diagram shows the effect of the small molecule compound RI-Q1 described in this invention on the degree of short-term myocardial ischemia-reperfusion injury in mice. A: Schematic diagram of the animal experiment process; BD: Evans blue and TTC double staining (B) and the corresponding percentage of infarct area to ischemic area (INF / AAV, C) and the ratio of myocardial ischemic area to left ventricle (AAR / LV, D) quantitative statistical results; EF: Tunel staining diagram of ischemic area of ​​myocardial tissue in each group of mice observed at 40× magnification (E) and the results of calculating the proportion of apoptotic cells in myocardium in each group (F); GI: Results of serum LDH (G), cTnT (H), and CK-MB (I) content determination.

[0022] Figure 5 This diagram illustrates the effect of the small molecule compound RI-Q1 described in this invention on the degree of myocardial ischemia-reperfusion injury in chronic mice. A: Schematic diagram of the animal experiment process; B: Gross specimen of mouse heart tissue stained with HE; CE: Location of fibrotic myocardial tissue using Masson staining (C), HE staining (D), and Sirius red staining (E); FG: Echocardiographic measurements of left ventricular ejection fraction (F) and left ventricular short axis shortening (G) in each group of mice; H: Echocardiographic results; I: Results of ANP content in mouse serum measured by ELISA. Detailed Implementation

[0023] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0024] This invention first demonstrates that conditional knockout of RIG-I in cardiomyocytes can effectively improve myocardial ischemia-reperfusion injury. Based on this, a potential inhibitor of the small-molecule RIG-I protein, RI-Q1, was obtained through virtual screening of existing commercial compound libraries. HL-1 mouse cardiomyocyte lines were cultured in a controlled hypoxic incubator with an O2 content controlled at 0.5% by replacing the air with a mixture of 95% N2 and 5% CO2 gas, and the culture medium was replaced with FBS and glucose-free DMEM for 4 hours. Subsequently, the culture medium was replaced with ordinary DMEM, and the cells were incubated under normoxic conditions (21% O2, 74% N2, and 5% CO2) for 6 hours before re-oxidation. A control group was cultured under normoxic conditions in FBS and glucose-deprived DMEM for the corresponding time to construct a cardiomyocyte hypoxia-reoxygenation model. The results showed that RI-Q1 significantly inhibited myocardial hypoxia-reoxygenation injury and effectively improved cell damage caused by pyroptosis. Subsequently, we utilized surface plasmon resonance (SPR) to perform biomolecular interaction analysis and protein pulldown techniques, confirming that RI-Q1 can directly bind to RIG-I protein. In a mouse model of myocardial ischemia-reperfusion injury, RI-Q1 effectively alleviated myocardial ischemia-reperfusion injury, reduced the infarct area, mitigated the decline in cardiac function caused by the injury, and reduced the release of downstream inflammatory factors. These results indicate that RI-Q1 specifically targets RIG-I to counteract the inflammatory effects induced by pyroptosis, thereby alleviating myocardial ischemia-reperfusion injury.

[0025] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0026] Laboratory animals: All animal care and experimental procedures used in this invention were approved by the Animal Policy and Welfare Committee of Wenzhou Medical University. For the short-term myocardial ischemia-reperfusion injury model mice, RI-Q1 (10 mg / kg / d, dissolved in DMSO to a 200 mmol / ml stock solution, then dissolved in 1% CMC-Na and 0-25% Tween-80) was administered intraperitoneally 2 hours before model establishment. For the long-term model, this formula was administered via gavage for a total of 14 days.

[0027] Experimental cells: The mouse HL-1 cells used in this invention were purchased from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The culture medium used was high-glucose DMEM (gibco, 11965092), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific). The cell culture environment was a 5% CO2 incubator at 37°C.

[0028] Example 1: Effect of conditional RIG-I knockout of cardiomyocytes on the severity of acute myocardial ischemia-reperfusion injury in mice This embodiment utilizes CRISPR / Cas9 technology to knock out exon 3, rendering RIG-I nonfunctional. The specific process was assisted by Cyagen Biosciences (https: / / www.cyagen.com / cn / zh-cn / sperm-bank-cko / 230073). RIG-I was then hybridized... fl / fl Myh6-Cre mice were used to obtain myocardial-specific RIG-I knockout mice. fl / fl Myh6-Cre + / - RIG-IcKO mice and RIG-Ifl / fl mice (RIGcKO-Sham) were used. RIG-IcKO-Sham and RIG-IcKO mice of similar age and weight were randomly selected by blind lottery and divided into four groups. Ischemia-reperfusion injury models were established according to the above protocol, with an ischemia time of 30 minutes and a reperfusion time of 24 hours. Figure 1 A). Evansblue and TTC double staining (TTC staining solution 2%, Solarbio, G3005) were used to compare the infarct and ischemic areas of the heart in mice of different groups, confirming that conditional RIG-I knockout of cardiomyocytes rescued cardiac function damage induced by acute myocardial ischemia-reperfusion. Figure 1 BD). After obtaining ischemic tissue from the myocardial regions of mice in each group, TUNEL staining was performed on frozen sections and observed under a 40× confocal microscope. The ratio of apoptotic cells in the ischemic myocardial regions of each group of mice was compared. The results confirmed that conditional knockout of RIG-I in cardiomyocytes effectively alleviated apoptosis in the process of myocardial ischemia-reperfusion injury. Figure 1 E, 1F). Additionally, we measured the levels of LDH, cTnT, and CK-MB in the serum supernatant of each group of mice (RIG-I). Based on these results, we found that the loss of RIG-I protein in cardiomyocytes effectively alleviated myocardial ischemia-reperfusion injury in mice; n = 6, p < 0.05 p<0.005, p<0.0001.

[0029] Example 2: High-throughput screening of RIG-I small molecule inhibitors to screen and verify the protective effect of RIG-I specific inhibitors.

[0030] ① Computer-aided drug design and virtual screening methods for high-throughput screening of RIG-I specific inhibitors First, using Glide v5.5 (Schrodinger Suite 2009) software, a computer-simulated screening of over 5 million small molecule compounds in existing commercial compound libraries was performed, targeting the published RIG-I-ATP-RNA ternary complex structure (PDB code: 3ZD7). The specific method is as follows: The Maestro v7.5 module in Glide software was used to remove all water molecules from the crystal structure and repair missing residues, followed by Glide molecular docking. First, the RIG-I protein was hydrogenated and charged using the "Protein Preparation" module, and optimized using the "Preparation and Refinement" option. To avoid spatial conflicts during docking, the local minimum search was terminated when the RMSD (Root-Mean-Square Deviation) reached its maximum value of 0.3 Å. The ATP binding site was defined as the docking center, the lattice size was 15 Å, and the van der Waals radii of atoms with a local charge less than 0.25 in the docking lattice were set to a scaling factor of 1. Molecular docking was performed on a Dell computer cluster server. Standard-Precision (SP) precision was then used to find the minimum energy conformation for compound binding to the RIG-I protein, and the Glide scoring mechanism (G-Score) was used to select the optimal three conformations for each compound. The top 2000 compounds in the virtual screening score ranking were then manually analyzed, and approximately 100 of the best potential drug candidates were purchased. Finally, the top 150 compounds were selected for subsequent pharmacological screening.

[0031] ② Inhibitor screening: Subsequently, a reporter gene screening system integrating IRF3, IRF7 and NF-κB promoters was constructed. Combined with computer-aided screening, 20 candidate compounds with significant inhibitory activity against RIG-I were screened out, as shown in Table 1.

[0032] Table 1

[0033] ③ In vitro validation of the effectiveness of small molecule compounds: In an experimental model of hypoxia-reoxygenation treatment stimulating cardiomyocytes, compounds such as D-16, D-44, D-45, D-54-1, and D-78 significantly improved cell viability. Plasmon resonance experiments further demonstrated that D-78 has the highest affinity for RIG-I; the candidate compound of D78 was named RI-Q1, and its chemical structure is shown below:

[0034] Example 3: This example investigates the protective effect of RI-Q1 on the classic hypoxia-reoxygenation model of the mouse cardiomyocyte line HL-1. In this invention, HL-1 cells were seeded into six-well plates and cultured in a 5% CO2, 37°C incubator until adherence and appropriate density were achieved. After washing three times with PBS, the cells were replaced with FBS and glucose-free DMEM. The cells were then cultured for 4 hours in a 0.5% O2, 5% CO2, 95% N2, 37°C hypoxic incubator. After replacing the FBS and high-glucose DMEM, the cells were cultured for another 6 hours in a standard 37°C incubator to construct a classic hypoxia-reperfusion (H / R) model. Figure 2 A). HL-1 cells were pretreated with RI-Q1 (10 μmol / ml, 20 μmol / ml, dissolved in DMSO) for 1 hour according to the grouping. Cell viability after H / R modeling was detected by CCK-8 (Cell Counting Kit-8) assay. Figure 2 B), the content of LDH released from the supernatant by cells was detected by LDH measurement to reflect changes in cell activity in each group ( Figure 2 (C) The results showed that RI-Q1 effectively improved cardiomyocyte activity after H / R treatment. Furthermore, morphological observation of cells in each group under a 10x light microscope showed that RI-Q1 reduced H / R-induced HL-1 cell shrinkage and death. Figure 2 D).

[0035] Example 4: RI-Q1 can directly bind to RIG-I protein and is kinetically stable. This invention uses AlphaFold to model the structure of human RIG-I (hRIG-I). After verifying the reliability of the hRIG-I model, the Caver web server for receptor tunneling and channel analysis is used to predict the binding sites of hRIG-I. Then, AutoDock 4.2 software is used to predict the binding mode between hRIG-I and RI-Q1. A cubic grid box with dimensions of 18.75 A × 18.75 A × 18.75 A is used to cover the binding sites of hRIG-I. SPR (Surface Plasmon Resonance) refers to the analysis of biomolecular interactions using Surface Plasmon Resonance. The Biacore™ molecular interaction analysis system, i.e., Cytiva's fully automated testing platform, is used to evaluate drug-protein binding relationships. This invention uses SPR to analyze the binding interaction between RI-Q1 and RIG-I proteins. RIG-I purified protein was diluted to 100 μg / mL with PBS at pH 5.5 and coupled to a blank CM5 chip in 10 mM sodium acetate buffer. RI-Q1 was serially diluted to seven concentrations with 1‰ DMSO + PBS, with the highest concentration being 40 μM and the lowest being 0.625 μM, for subsequent detection. The samples were added sequentially to the sample plate, and the program was run. Kinetic and steady-state data fitting were performed using Biacore™ software. Finally, the obtained data were fitted using a 1:1 fitting model. SPR results showed that RIG-I protein and RI-Q1 exhibited kinetic stability, with a KD value of 6.12e-05M (…). Figure 3 A).

[0036] In addition, this invention uses Beaverbeads™ streptomycin affinity-agarose beads and biotinylated RI-Q1 to perform protein pull-down experiments, verifying the binding of RIG-I and RI-Q1 in purified RIG-I protein, HL-1 cell line, ischemic myocardial tissue after PCI in patients with ischemic cardiomyopathy, and ischemic-reperfusion heart tissue in mice. Biotinylated RI-Q1 was added to streptomycin affinity-agarose beads at a ratio of 10:1, with untreated RI-Q1, biotin alone, and untreated streptomycin affinity-agarose beads serving as control groups. Then, purified RIG-I protein was added to streptomycin affinity-agarose beads containing Bio-RI-Q1. The mixture was incubated at 25°C for 3 hours on a shaker, centrifuged, and the precipitate was washed three times. Finally, the protein was eluted with 5x loading solution by boiling the precipitate to obtain a RIG-I protein sample bound to RI-Q1. Samples were grouped and analyzed by Western blotting. Samples were loaded onto polyacrylamide gels for electrophoresis, and detected using RIG-I primary antibody and corresponding species-specific secondary antibody. Color development was performed using ultrasensitive exposure buffer. Figure 3 BE). Based on the results of SPR and protein pulldown experiments, it was found that RI-Q1 directly binds to the purified RIG-I protein. Figure 3 BE).

[0037] Example 5: Improvement of the severity of acute myocardial ischemia-reperfusion injury in mice by RI-Q1 Animal level: Healthy, age-matched (8 weeks old) C57BL / 6 mice of similar weight were randomly divided into five groups of 10 mice each by blind selection. Each group received the corresponding dose of RI-Q1 (10 mg / kg, 20 mg / kg) before surgery according to the above protocol. Figure 4 A). The ratio of infarct area to ischemic area in the heart of each group of mice was calculated by Evans blue and TTC double staining (TTC staining solution 2%, Solarbio, G3005). Figure 4 BD), and the proportion of apoptotic myocardial cells in each group was calculated at 40× magnification using TUNEL apoptosis staining. Figure 4 EF). In addition, the serum levels of LDH, cTnT, and CK-MB in each group of mice were measured. Figure 4 (GI), combined with the above results, it was found that RI-Q1 can significantly reduce myocardial ischemia-reperfusion injury in mice. n = 6, p < 0.05 p<0.005, p<0.0001.

[0038] Example 6: Small molecule compound RI-Q1 improves chronic myocardial ischemia-reperfusion injury in mice Healthy, age-matched (8 weeks) C57BL / 6 mice of similar weight were randomly divided into five groups of 12 mice each by blind selection. For two weeks prior to modeling, each group received a daily intragastric gavage of 10 mg / kg / day (DMSO dissolved in 200 mmol / ml stock solution, then dissolved in 1% CMC-Na and 0-25% Tween-80). Under the support of a small animal ventilator and gas anesthesia machine, the thoracic cavity was surgically exposed, and the left anterior descending artery was ligated with 7-0 absorbable sutures. The sutures were released after 30 minutes, and the successful establishment of the myocardial ischemia-reperfusion injury model was confirmed by visually observing the return of pale myocardium to redness. The control group underwent a sham surgery in the same manner. After modeling, the mice were housed in a constant temperature and humidity SPF environment at 37°C. Surgery, the mice in each group were administered the above-mentioned intragastric gavage every other day for two weeks. Figure 5 A). Left ventricular ejection fraction and left ventricular fractional shortening were calculated in each group of mice using echocardiography. Figure 5 FH), and the results showed that RI-Q1 could alleviate cardiac function decline caused by myocardial ischemia-reperfusion injury. After euthanizing mice with an overdose of isoflurane, we collected perfused heart tissue and serum. Gross longitudinal sections of mouse heart tissue were stained with hematoxylin and eosin (HE) to visualize the entire long axis section of the heart, including the cardiac chambers, myocardium, epicardium, and other overall structures. Figure 5 B), and by localizing fibrous tissue using Masson's staining, HE staining, and Sirius red staining, the results showed that RI-Q1 can alleviate myocardial fibrosis caused by myocardial ischemia-reperfusion injury (B). Figure 5 CE). Similarly, the determination of ANP levels in the serum of mice in each group also confirmed this result. Figure 5 I).

[0039] The method for preparing a drug for treating or preventing myocardial ischemia-reperfusion injury provided in this invention includes: various dosage forms of the pharmaceutical composition of this invention can be prepared according to conventional pharmaceutical manufacturing methods. For example, the active ingredient RI-Q1 is mixed with one or more carrier excipients, and then formulated into the desired dosage form, such as injections, capsules, and tablets.

[0040] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of RIG-I as a detection target in screening drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

2. Application of RIG-I inhibitors in the preparation of drugs for the prevention and / or treatment of myocardial ischemia-reperfusion injury.

3. The application according to claim 2, characterized in that, The RIG-I inhibitors include: i) any one or more of shRNA, siRNA, ASO, and delivery vectors of said shRNA, siRNA, and ASO, as well as reagents of the CRISPR / Cas system and the Cre-loxP system that specifically inhibit RIG-I expression in myocardial tissue or cardiomyocytes; ii) any one or more of neutralizing antibodies, aptamers, and small molecule inhibitors that specifically bind to RIG-I.

4. The application according to claim 3, characterized in that, The delivery vector may be a viral vector or a non-viral vector.

5. The application according to claim 4, characterized in that, The viral vector is selected from one or more combinations of retroviral vectors, adenovirus vectors, and adeno-associated virus vectors; and / or, the non-viral vector includes mRNA liposomes.

6. The application according to claim 3, characterized in that, The RIG-I inhibitor is a reagent (including Cre recombinase) required for conditional knockout of RIG-I in cardiomyocytes via the Cre-loxP system.

7. The application according to claim 3, characterized in that, The RIG-I inhibitor is a small molecule compound RI-Q1, with the molecular formula: C 12 H 10 ClN3O3S, chemical structural formula is:

8. The application according to claim 3, characterized in that, The drug comprises an active ingredient and pharmaceutically acceptable excipients, the active ingredient including a RIG-I inhibitor.

9. The application according to claim 3, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: diluents, binders, wetting agents, lubricants, disintegrants, solvents, emulsifiers, cosolvents, preservatives, pH adjusters, osmotic pressure adjusters, surfactants, coating materials, antioxidants, and buffers. And / or, the dosage form of the drug includes any one of tablets, pills, suspensions, granules, and emulsions.

10. A RIG-I inhibitor with anti-pyroptosis activity, characterized in that, The RIG-I inhibitor is formulated from RI-Q1 and pharmaceutically acceptable excipients.

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