Application of CGAS-STING signal channel inhibitor in preparation of medicine for preventing and treating hepatic ischemia-reperfusion injury

Abstract

The invention discloses application of a CGAS-STING signal channel inhibitor in preparation of a medicine for preventing and treating hepatic ischemia-reperfusion injury, and relates to the technical field of biological medicine. According to the application, a cGAS-STING signal channel in macrophages is inhibited by using a cGAS inhibitor or an STING inhibitor, activation of double-stranded deoxyribonucleic acid released by a neutrophile granulocyte extracellular trapping net on a DNA induction channel is blocked, and the M1 type polarization level of the macrophages is reduced. The traditional Chinese medicine composition can effectively relieve liver inflammatory response and cell apoptosis, and a new path is provided for preventing and treating acute liver injury after liver transplantation or liver resection.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of a cGAS-STING signaling pathway inhibitor in the preparation of drugs for preventing and treating liver ischemia-reperfusion injury. Background Technology

[0002] Hepatic ischemia-reperfusion injury is an unavoidable pathophysiological process during hepatectomy and liver transplantation, and a major factor leading to postoperative graft dysfunction and biliary complications. This process is generally divided into an ischemic phase and a reperfusion phase: the ischemic phase leads to disruption of the mitochondrial electron transport chain and accumulation of abnormal metabolites; the reperfusion phase, with the restoration of blood flow, triggers a burst of reactive oxygen species, thereby activating a strong aseptic inflammatory response. This inflammatory response leads to hepatocyte necrosis, apoptosis, or ferroptosis.

[0003] Among existing clinical intervention strategies, mechanical perfusion is a core approach to reducing graft injury. For example, hypothermic oxygenation perfusion reduces oxidative damage by repairing mitochondrial metabolism, while normothermic mechanical perfusion is used to simulate the physiological environment to assess organ function. In addition, adjunctive methods such as antioxidants and ischemic preconditioning are frequently studied in clinical practice. However, even with these interventions, the inflammatory cascade response triggered by injury-related molecular patterns remains difficult to completely block.

[0004] Studies have shown that intrinsic macrophages in the liver polarize towards a pro-inflammatory M1 phenotype after being stimulated by injury signals, and these polarized macrophages are major effectors of tissue destruction. Although it is known that neutrophils are recruited to the damaged liver and release neutrophil extracellular traps to participate in the tissue destruction process, accurately identifying and blocking signal crosstalk between neutrophils and macrophages remains a research challenge. Currently, the key molecular pathways that precisely link upstream neutrophil activation with downstream macrophage pro-inflammatory polarization are unclear, resulting in a lack of molecularly targeted therapies that can specifically interrupt this pathogenic cascade in clinical practice. Therefore, there is an urgent need for a technical solution that can effectively intervene in this inflammatory axis to more precisely alleviate hepatic ischemia-reperfusion injury. Summary of the Invention

[0005] This invention provides the use of cGAS-STING signaling pathway inhibitors in the preparation of drugs for preventing and treating liver ischemia-reperfusion injury, solving the problems of existing technologies being unable to completely block inflammatory cascade reactions and lacking targeted therapies against immune crosstalk.

[0006] This invention proposes the use of a cGAS-STING signaling pathway inhibitor in the preparation of drugs for preventing and treating liver ischemia-reperfusion injury. The cGAS-STING signaling pathway inhibitor is either a cGAS inhibitor or a STING inhibitor. The drug uses a cGAS inhibitor or a STING inhibitor as its active ingredient. Liver ischemia-reperfusion injury is characterized by the activation of the cGAS-STING signaling pathway in macrophages by double-stranded DNA released from the extracellular trap of neutrophils, which induces macrophages to M1 polarization, thereby aggravating the pathological feature of liver tissue damage. The drug reduces liver inflammation and apoptosis by inhibiting the cGAS-STING signaling pathway in macrophages and downregulating the M1 polarization level of macrophages.

[0007] Preferably, the cGAS inhibitor is RU.521.

[0008] Preferably, the drug further includes a neutrophil extracellular trap inhibitor, wherein the neutrophil extracellular trap inhibitor is deoxyribonuclease I.

[0009] Preferably, hepatic ischemia-reperfusion injury occurs during liver transplantation or liver resection.

[0010] Preferably, downregulating the M1 polarization level of macrophages refers to reducing the expression level of one or more of the pro-inflammatory markers inducible nitric oxide synthase, interleukin-6, tumor necrosis factor-α, or interleukin-1β in macrophages.

[0011] Preferably, inhibiting the cGAS-STING signaling pathway in macrophages means reducing the expression level of cGAS protein in macrophages, or reducing the phosphorylation level of one or more of the following proteins: STING protein, TBK1 protein, or IRF3 protein.

[0012] Preferably, in the intended use, the drug is prepared as an injectable formulation suitable for intraperitoneal or intravenous injection.

[0013] Preferably, the drug is a combination formulation of a cGAS inhibitor or STING inhibitor and deoxyribonuclease I as the active ingredient, used to produce a dual inhibitory effect of degrading extracellular neutrophil extracellular traps and blocking intracellular double-stranded DNA sensing.

[0014] Preferably, the drug is used to alleviate acute liver injury within 12 hours after hepatic ischemia-reperfusion, characterized by coagulative necrosis of hepatocytes, congestion of hepatic sinusoidal vessels, and elevated levels of serum transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0015] Preferably, the macrophages are intrinsic liver macrophages, Kuff cells, or macrophages differentiated from peripheral blood differentiation cluster 14 positive monocytes; the neutrophil extracellular trap is a double-stranded DNA network structure containing citrullinated histone H3, myeloperoxidase, and neutrophil elastase released by activated neutrophils.

[0016] Beneficial Effects: This invention provides the use of an inhibitor of the cyclic guanosine monophosphate (cGMP)-adenosine monophosphate (AAMP)-interferon gene stimulating factor (IRGF) signaling pathway in the preparation of drugs for preventing and treating liver ischemia-reperfusion injury. By blocking the DNA sensing pathway activated by double-stranded deoxyribonucleic acid (DDNA) derived from neutrophil extracellular traps in macrophages, it achieves precise regulation of the liver's inflammatory microenvironment. The inhibitor described in this invention can effectively block NETs-mediated macrophage M1 polarization, disrupting the inflammatory cascade at the molecular level and significantly reducing the expression of pro-inflammatory factors such as inducible nitric oxide synthase (MODS) in liver tissue. This invention significantly reduces coagulative necrosis and apoptosis of hepatocytes, resulting in a substantial decrease in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, providing a clear targeted intervention pathway for alleviating early graft dysfunction in liver transplantation. Attached Figure Description

[0017] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is a diagram illustrating the formation and related analysis of extracellular trapping networks of neutrophils in the peripheral blood of liver transplant recipients; among them, Figure 1 A represents the differential gene volcano diagram. Figure 1 B is a Venn diagram showing the intersection of differentially expressed genes and the gene set of the neutrophil extracellular trap. Figure 1 C is a bar chart showing the extracellular trapping net score of neutrophils in each group of samples. Figure 1 D and Figure 1 E is an immunofluorescence staining image. Figure 1 F is a graph showing serum double-stranded deoxyribonucleic acid (DDNA) content. Figure 1 G is a correlation graph of serum alanine aminotransferase (ALT) levels.

[0018] Figure 2 This is a time-dynamic graph showing the deposition of extracellular traps of neutrophils in a mouse model of liver ischemia-reperfusion injury; among which, Figure 2 A shows a hematoxylin-eosin staining image of liver tissue. Figure 2 B is the apoptosis detection graph. Figure 2 C represents the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Figure 2 D represents the serum double-stranded deoxyribonucleic acid (DDNA) level graph. Figure 2 E shows the immunofluorescence staining and intensity analysis of the extracellular trapping network components of neutrophils in liver tissue.

[0019] Figure 3 This is a verification diagram showing how neutrophil extracellular traps induce macrophage polarization towards the M1 type; among them, Figure 3 A is a heatmap of immune cell infiltration. Figure 3 B is the correlation analysis plot. Figure 3 C represents the protein blot images of polarization markers at different reperfusion time points. Figure 3 D is a Western blot image of polarization markers after the application of deoxyribonuclease I. Figure 3 E is a Western blot of polarization indices from an in vitro co-culture model. Figure 3 F is a flow cytometry image of macrophage surface markers.

[0020] Figure 4 This is a verification diagram showing how neutrophil extracellular traps induce macrophage polarization through direct interactions; among them, Figure 4 A is a diagram illustrating direct and indirect co-culture models. Figure 4 B is an immunofluorescence confocal image of macrophages taking up components of the neutrophil extracellular trap. Figure 4 C represents the protein blot diagrams of polarization markers under different co-culture modes.

[0021] Figure 5 This is a verification diagram of the pathway activating macrophage cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene stimulatory factor by the extracellular trapping network of neutrophils; among which, Figure 5 A is a diagram showing the differences in signal pathways. Figure 5 B shows the Western blot diagrams of proteins related to the signaling pathway at different reperfusion time points. Figure 5 C is a Western blot map of signaling pathway proteins in an in vitro co-culture model. Figure 5 D is an enzyme-linked immunosorbent assay (ELISA) graph showing the content of pro-inflammatory factors.

[0022] Figure 6 This is a verification diagram showing the pharmacological inhibition of neutrophil extracellular traps or cyclic guanylate-adenosine monophosphate synthase to reduce liver damage; among them, Figure 6 A is a Western blot image of polarization markers in an in vitro inhibition experiment. Figure 6 B is a Western blot image of the pathway protein in the in vitro inhibition experiment. Figure 6 C is the Western blot diagram of polarization markers after intervention in the mouse model. Figure 6 D is a Western blot map of pathway proteins after intervention in the mouse model. Figure 6 E shows a diagram of liver tissue pathological staining and apoptosis detection. Figure 6 F is a graph showing the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice. Detailed Implementation

[0023] To more clearly illustrate the technical solutions in the embodiments of the invention or the prior art, the invention will be briefly introduced below in conjunction with the accompanying drawings and descriptions of the embodiments or the prior art. Obviously, the following description of the structure of the drawings is only some embodiments of the invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the description of these embodiments is for the purpose of helping to understand the invention, but does not constitute a limitation on the invention.

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments, but it is not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments of the present invention are conventional methods; the materials and reagents used, unless otherwise specified, can be obtained commercially.

[0025] The experimental animals involved in this invention embodiment are male Cluster of Differentiation 57 (C57BL / 6) mice, aged 8 to 12 weeks and weighing between 20 and 25 grams, purchased from a professional experimental animal center. The animals were housed in a standard-grade experimental animal facility with a constant temperature of 22 to 24 degrees Celsius, humidity of 50% to 60%, and a regular diurnal rhythm, with free access to water and food. The main experimental reagents included: Deoxyribonuclease I (DNase I), with an activity of ≥2000 units per mg, purchased from Solarbio Science & Technology Co., Ltd.; Cyclic GMP-AMP Synthase (cGAS) inhibitor RU.521, with a chemical purity of ≥98%, purchased from Corning Life Sciences Co., Ltd.; Phorbol 12-Myristate13-Acetate (PMA), purchased from Sigma-Aldrich; Fetal bovine serum and RPMI 1640 medium, both sterilized, purchased from Gibbs & Co.; Recombinant Human Macrophage Colony-Stimulating Factor (rhM-CSF), with a purity of ≥95%, purchased from Pipertec; Anti-differentiation Cluster 14 magnetic bead system, purchased from Miltenyi Biotechnology Co., Ltd.; and Double-Stranded Deoxyribonucleic Acid (DDNA). The Acid (dsDNA) detection kit, using a high-sensitivity fluorescence method, was purchased from Thermo Fisher Scientific. The antibodies used for Western blotting and immunofluorescence included anti-myeloperoxidase (MPO), anti-neutrophil elastase (NE), anti-citrullinated histone H3 (CitH3), anti-cGAS, anti-stimulator of interferon genes (STING), anti-phosphorylated interferon gene stimulator, anti-inducible nitric oxide synthase (iNOS), and anti-Cluster of Differentiation 206 (CD206). The antibody dilution was a phosphate-buffered saline solution containing 1% bovine serum albumin.

[0026] In an important embodiment of this invention, the isolation of human peripheral blood neutrophils and the induction and quantification of neutrophil extracellular traps (NETs) are first performed. Using a human bone marrow neutrophil separation kit, neutrophils are isolated from healthy donor peripheral blood via a three-layer density gradient centrifugation method. Specifically, heparin-anticoagulated whole blood is slowly layered onto the top layer of the density gradient solution, and centrifuged at 400g for 30 minutes at 20°C with acceleration set to level 5 and deceleration set to level 0 to prevent agitation of the interface. The middle white granular cell layer is carefully aspirated and washed twice with sterile phosphate-buffered saline, centrifuged at 300g for 10 minutes each time. The isolated neutrophils are resuspended in phenol red-free RPMI 1640 medium. The cell concentration is adjusted to 3 x 10⁶ cells / mL using an automated cell counter or hemocytometer. Purity is confirmed by flow cytometry to detect differentiation cluster 11b and Ly6G markers, with a purity greater than 95%. To induce NET formation, PMA was added to a neutrophil suspension at a final concentration of 100 nanomoles per liter, and the suspension was incubated for 4 hours in a humidified incubator at 37°C and 5% CO2. After incubation, the supernatant containing cell debris and stimulants was gently aspirated, and the bottom of the wells was washed with pre-cooled calcium- and magnesium-free Hanks' balanced salt solution. The collected suspension was centrifuged at 4°C and 400g for 5 minutes to completely remove unruptured cells. The resulting supernatant was the purified NET suspension. The dsDNA content was measured using a dsDNA detection kit with PicoGreen fluorescence staining, and adjusted to a uniform concentration of 5 micrograms per milliliter for subsequent experiments.

[0027] Subsequently, an induction differentiation experiment was conducted on macrophages derived from human peripheral blood. Approximately 20 ml of fresh peripheral blood was collected from the subjects and centrifuged at 20°C and 400g for 25 minutes using the Ficoll density gradient centrifugation method to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were resuspended in centrifugation buffer containing Cluster of Differentiation 14 (CD14) magnetic beads and incubated at 4°C in the dark for 15 minutes. Subsequently, CD14-positive mononuclear cells were screened using an LS sorting column placed in a magnetic field. The sorted mononuclear cells were seeded in RPMI 1640 medium containing 10% inactivated fetal bovine serum and 50 ng / mL rhM-CSF, with an initial seeding density of 106 cells / mL, and cultured in 6-well plates. The plates were continuously cultured at 37°C for 7 days, with half-volume medium replacement performed on days 3 and 5 to maintain nutrient supply. By day 7, under an inverted phase-contrast microscope, the cells exhibited a typical macrophage pseudopodia-like adherent morphology, indicating differentiation into mature resting macrophages, commonly referred to as M0 macrophages.

[0028] In animal experiments, this invention established a mature mouse model of liver ischemia-reperfusion injury. Mice were fasted for 12 hours preoperatively to ensure a clear surgical field and prevent aspiration. Anesthesia was administered via intraperitoneal injection of a 1.25% tribromoethanol solution at a dose of 125 mg per kilogram of body weight. During surgery, a midline abdominal incision of approximately 2 cm was made using ophthalmic scissors. The liver and hepatic pedicle were exposed, and the hepatic hilum was carefully dissected under a stereoscopic operating microscope. The three-way junction supplying the left and middle lobes of the liver was accurately clamped using non-invasive microvascular clamps. Ischemia time was precisely controlled to 60 minutes using a stopwatch. During this time, preheated saline gauze was used to cover the abdominal incision to prevent fluid loss and hypothermia. During the reperfusion phase, the vascular clamps were removed, and the liver color was observed to return from pale to pink within 30 seconds, confirming unobstructed blood flow. Subsequently, continuous suturing of the muscle and skin layers was performed using 5-0 sterile sutures. Serum samples from the inferior vena cava and liver tissue samples from the corresponding damaged sites were collected from mice at 6, 12, and 24 hours after reperfusion. Mice in the sham-operated group underwent the same laparotomy and hepatic hilum dissection procedures, but without clamping the blood vessels. Serum samples were centrifuged and stored at -80°C. A portion of the liver tissue was fixed in 4% paraformaldehyde solution for 24 hours for sectioning, while the other portion was immediately flash-frozen in liquid nitrogen for Western blotting experiments.

[0029] In vitro cell co-culture experiments were used to investigate the mechanisms of cell-cell interactions. In the direct co-culture model, the aforementioned purified NETs were directly added to a culture system of differentiated M0 macrophages at a final concentration of 5 μg / mL dsDNA, and co-cultured for 12 hours. In the indirect co-culture model, a 0.4 μm transwell culture system was used. Macrophages were seeded in the lower chamber, and NETs were added to the upper chamber, utilizing a semi-permeable membrane physical barrier to prevent direct physical contact between the NETs and the macrophages in the lower chamber. After co-culture, cells were collected for protein and ribonucleic acid extraction. In the inhibition experiment, macrophages were pretreated with RU.521 at a final concentration of 2.5 μg / mL one hour before the addition of NETs.

[0030] Embodiment 1 of this invention details the application value of cGAS-STING signaling pathway inhibitors in preventing and treating liver ischemia-reperfusion injury. For example... Figure 1 A and Figure 1 As shown in B, the transcriptomic dataset GSE14951 for liver ischemia-reperfusion was first screened using bioinformatics methods. Analysis revealed a high degree of overlap between differentially expressed genes and gene sets associated with NET formation. In clinical sample validation, such as... Figure 1 D and Figure 1 As shown in Figure E, immunofluorescence co-localization technology revealed a large number of reticular structures composed of CitH3, NE, and MPO in the peripheral blood of liver transplant recipients within 24 hours post-surgery, confirming the massive release of NETs in vivo. Figure 1 As shown in Figure F, the level of dsDNA in the recipient's serum was significantly elevated, and as... Figure 1 As shown in G, this level showed a very strong positive correlation with the level of alanine aminotransferase (ALT), which reflects the degree of liver damage, with a correlation coefficient r greater than 0.8.

[0031] Dynamic observations in a mouse model further confirmed the aforementioned pathological process. Figure 2 A and Figure 2 As shown in Figure B, the 12-hour reperfusion period is the most severe time for liver tissue damage. Hematoxylin-Eosin (H&E) staining reveals large areas of sheet-like coagulative necrosis foci. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) detection shows dense apoptosis-positive signals in the damaged liver. Figure 2As shown in Figure C, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities reach their peak at this point. Figure 2 D and Figure 2 The high synchronization of NET deposition in the liver, as shown in Figure E, indicates that the first 12 hours after reperfusion are a critical phase for NET-driven inflammatory damage. Figure 3 A and Figure 3 As shown in Figure B, thermographic analysis of immune cell infiltration revealed a significant polarization of hepatic macrophages towards the pro-inflammatory M1 phenotype. Figure 3 As shown in Figure C, Western blot results confirmed that the protein bands of the M1 polarization marker iNOS and interleukin-6 (IL-6) were strongest at 12 hours. This occurred after in vivo administration of DNase I to degrade NETs ( Figure 3 D), the above polarization indices decreased significantly. In vitro experiments ( Figure 3 E and Figure 3 F) also showed that NETs stimulation could significantly induce high expression of Cluster of Differentiation 86 (CD86) on the surface of macrophages.

[0032] More crucial mechanism discovery, such as Figure 4 A, Figure 4 B and Figure 4 As shown in Figure C, transwell culture experiments demonstrated that macrophages can only initiate polarization by recognizing and taking up NET structures under direct physical contact. Fluorescence microscopy clearly captured CitH3-positive NET fragments encapsulated in the macrophage cytoplasm. This dependence on direct contact reveals the crucial role of the intracytoplasmic DNA sensing pathway. Figure 5 A to Figure 5 As shown in Figure D, this invention confirms that the polarization effect induced by NETs is entirely dependent on the activation of the cGAS-STING signaling pathway. At 12 hours of reperfusion, the phosphorylation of STING protein, tank-binding kinase 1 (TBK1) protein, and interferon regulatory factor 3 (IRF3) protein in liver tissue significantly increased. Furthermore, in an in vitro model, NETs were observed to rapidly trigger this phosphorylation signaling pathway, leading to a burst of release of pro-inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Figure 5 D).

[0033] The therapeutic potential of this invention was ultimately verified through pharmacological inhibition experiments. Figure 6 A to Figure 6 As shown in Figure F, after applying the cGAS-specific inhibitor RU.521, in both in vitro cell models and whole-animal models, even though NETs stimulation was still present in the liver, downstream STING phosphorylation was completely inhibited due to the blockade of cGAS's sensing of dsDNA. Figure 6 E and Figure 6 As shown in Figure F, the necrotic area in the mouse liver was significantly reduced, serum transaminase levels decreased by more than 50%, and macrophages maintained a low M1 polarization state. This series of detailed experimental data indicates that cGAS-STING signaling pathway inhibitors can effectively protect the liver from acute reperfusion injury by specifically disrupting the communication chain between NETs and macrophage polarization.

[0034] In specific implementations, this invention can also provide the following preferred implementation paths for different application scenarios. Preferably, the cGAS inhibitor is RU.521, whose mechanism of action is to competitively block the catalytic site of cGAS, thereby preventing the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) at the signal source. Preferably, the drug for preventing liver ischemia-reperfusion injury can also be designed as a combination formulation, that is, combining the aforementioned pathway inhibitor with deoxyribonuclease I. The synergistic advantage of this combination is that deoxyribonuclease I is responsible for clearing the large molecular structures of NETs accumulated in the extracellular space, reducing the pro-inflammatory load in the microcirculation; while the cGAS inhibitor is responsible for blocking DNA residues that have crossed the macrophage barrier and entered the cytoplasm. The synergistic effect of both achieves dual targeting of the source and internalization of inflammatory signals.

[0035] Preferably, the prevention and treatment of hepatic ischemia-reperfusion injury is specifically targeted at liver transplantation or major hepatectomy. During clinical liver transplantation, the explosive infiltration of neutrophils during reperfusion after prolonged cryopreservation of the donor organ is considered a major pathological basis for graft injury. The application provided by this invention demonstrates excellent organ protection capabilities within this time window. Preferably, downregulating macrophage M1 polarization levels specifically manifests as reducing the protein expression levels of one or more pro-inflammatory factors among inducible nitric oxide synthase, interleukin-6, tumor necrosis factor-α, or interleukin-1β. These factors are recognized as key toxic mediators leading to hepatic microvascular microembolism and widespread hepatocyte necrosis. Preferably, the molecular evaluation marker for inhibiting the cGAS-STING signaling pathway is a significant inhibition of the phosphorylation activation state of STING protein, tank-binding kinase 1 protein, or interferon regulatory factor 3 protein, which provides standardized, molecular-level quantitative indicators for preclinical drug screening and efficacy evaluation.

[0036] Preferably, targeting the acute kinetic window of hepatic ischemia-reperfusion injury development, the drug used in this invention is prepared as an injectable formulation suitable for intraperitoneal or intravenous injection, and its carrier may include physiological saline, phosphate buffer, or other biocompatible solvents. In clinical practice, the administration time is preferably 30 minutes before vascular clamping or immediately after reperfusion to ensure that the active ingredient reaches an effective concentration in liver tissue before the peak of pathological damage. Preferably, the macrophages targeted by the drug of this invention include both Kupffer cells in situ in the liver and exogenous macrophages recruited and differentiated from peripheral blood cluster 14 positive monocytes after reperfusion. As the primary regulator of the hepatic immune microenvironment, the sensitivity of Kupffer cells to cGAS-STING signaling determines the intensity of the overall inflammatory cascade. Finally, preferably, this drug is specifically designed to alleviate acute liver tissue disintegration within 12 hours after reperfusion, with objective evaluation endpoints being a reduction in hepatocyte necrosis area and a significant downregulation of serum transaminase activity.

[0037] To further ensure the feasibility of this invention, more detailed experimental parameters are provided below. In the Western blot experiment, a polyvinylidene fluoride (PVDF) membrane with a pore size of 0.45 μm was used. The blocking solution was a phosphate buffer solution containing 5% skim milk powder. The primary antibody was incubated overnight at 4°C on a shaker, and the secondary antibody was incubated at room temperature for 1 hour. Development was performed using an enhanced chemiluminescence (ECL) system. Data quantification was performed using ImageJ software for image analysis. For statistical processing, all data are expressed as mean plus or minus standard deviation. Statistical calculations were performed using GraphPad Prism 9.0 software. Unpaired Student's t-tests were used for comparisons between two groups. One-way ANOVA combined with Tukey's post-test was used for comparisons among multiple groups. All p-values ​​were based on two-tailed tests, and a p-value less than 0.05 was considered statistically significant. Through the detailed experimental steps and multi-dimensional parameter limitations described above, those skilled in the art can completely replicate and implement the prevention and control scheme of the present invention.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0039] Finally, it should be noted that although embodiments of the present invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. The use of a cGAS-STING signaling pathway inhibitor in the preparation of a medicament for preventing and treating liver ischemia-reperfusion injury, characterized in that, The cGAS-STING signaling pathway inhibitor is a cGAS inhibitor or a STING inhibitor; the drug takes the cGAS inhibitor or the STING inhibitor as an active ingredient; the liver ischemia-reperfusion injury has the pathological characteristics of activating the cGAS-STING signaling pathway in macrophages by double-stranded DNA released from neutrophil extracellular traps, and inducing the M1 polarization of the macrophages to aggravate the liver tissue injury; The drug down-regulates the M1 polarization level of the macrophages by inhibiting the cGAS-STING signaling pathway in the macrophages, so as to reduce the inflammatory response and apoptosis of the liver.

2. Use according to claim 1, characterized in that, The cGAS inhibitor is RU.

521.

3. Use according to claim 1, characterized in that, The drug further comprises a neutrophil extracellular trap inhibitor, and the neutrophil extracellular trap inhibitor is DNase I.

4. Use according to claim 1, characterized in that, The liver ischemia-reperfusion injury occurs in a liver transplantation surgery or a liver resection surgery.

5. Use according to claim 1, characterized in that, The down-regulation of the M1 polarization level of the macrophages refers to the reduction of the expression level of one or more of inducible nitric oxide synthase, interleukin-6, tumor necrosis factor-α, or interleukin-1β in the macrophages.

6. Use according to claim 1, characterized in that, The inhibition of the cGAS-STING signaling pathway in the macrophages refers to the reduction of the expression level of cGAS protein in the macrophages, or the reduction of the phosphorylation level of one or more of STING protein, TBK1 protein, or IRF3 protein.

7. Use according to claim 1, characterized in that, In the use, the drug is prepared into an injection preparation suitable for intraperitoneal injection or intravenous injection.

8. Use according to claim 3, characterized in that, The drug takes the cGAS inhibitor or the STING inhibitor, and a composition preparation taking the DNase I as an active ingredient, to produce a double inhibition effect of degrading extracellular neutrophil extracellular traps and blocking intracellular double-stranded DNA sensing.

9. Use according to claim 1, characterized in that, The drug is used for reducing acute liver injury within 12 hours after liver ischemia-reperfusion, and the acute liver injury has the pathological characteristics of hepatocyte coagulative necrosis, liver sinusoidal vessel hyperemia, and the increase of serum transaminase glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase levels.

10. Use according to claim 1, characterized in that, The macrophages are Kupffer cells in the liver or macrophages differentiated from peripheral blood cluster of differentiation 14 positive mononuclear cells; and the neutrophil extracellular traps are a double-stranded DNA network structure containing citrullinated histone H3, myeloperoxidase, and neutrophil elastase released by activated neutrophils.

Images