Method for improving ischemia reperfusion injury by inhibiting TRIM25

Abstract

The invention relates to the technical field of biological medicines, and discloses a method for improving ischemia-reperfusion injury by inhibiting TRIM25. According to the method, expression of TRIM25 or interaction between TRIM25 and PGAM5 is inhibited, a TRIM25-siRNA drug delivery system containing a specific sequence or a TRIM25-PGAM5 interaction blocking peptide is inhibited, mtDNA release-cGAS-STING is cut off, and activation of NLRP3 inflammation bodies is blocked, so that the ischemia-reperfusion injury is improved. Tissue damage and inflammatory cell death are relieved; according to the invention, the TRIM25-siRNA drug delivery system is orally taken 24 hours before an operation, and the TRIM25-PGAM5 interaction blocking peptide is injected in an intraperitoneal cavity at the beginning of perfusion.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method for improving ischemia-reperfusion injury by inhibiting TRIM25. Background Technology

[0002] Ischemia-reperfusion injury (IRI) is a common clinical pathological process, referring to the exacerbation of damage to ischemic tissue after blood flow is restored. Its core mechanisms are closely related to mitochondrial dysfunction, excessive inflammatory response, and pyroptosis. Intestinal ischemia-reperfusion injury (II / R) is a typical example, which can lead to intestinal barrier disruption, bacterial translocation, and even systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). Currently, there are no effective clinical treatments.

[0003] Studies have confirmed that TRIM25 is significantly upregulated in IRI, increasing mitochondrial membrane permeability and promoting the release of mitochondrial DNA (mtDNA) into the cytoplasm through direct interaction with the mitochondrial outer membrane phosphatase PGAM5. The released mtDNA is recognized by cGAS and activates the cGAS-STING pathway, thereby triggering NLRP3 inflammasome assembly and pyroptosis, ultimately exacerbating tissue damage. Therefore, inhibiting TRIM25 can block the aforementioned pathological signaling pathways, providing a new target for IRI treatment.

[0004] Therefore, providing a method to improve ischemia-reperfusion injury by targeting and inhibiting TRIM25 is of great practical significance. Summary of the Invention

[0005] In view of this, the present invention proposes a method to improve ischemia-reperfusion injury by inhibiting TRIM25, aiming to solve the problems in the current technology of ischemia-reperfusion injury treatment, such as the lack of effective intervention for II / R, poor non-targeted treatment with significant side effects, insufficient stability and targeting, and inability to cover the needs of different clinical scenarios.

[0006] This invention proposes a method to improve ischemia-reperfusion injury by inhibiting TRIM25, characterized in that the method includes blocking downstream mitochondrial damage, mtDNA release and inflammatory pathway activation by inhibiting the expression of TRIM25 or its interaction with PGAM5; The inhibition of TRIM25 refers to the administration of a TRIM25-targeting inhibitor, which inhibits TRIM25 expression or blocks the interaction between TRIM25 and PGAM5 by inhibiting the TRIM25-siRNA drug delivery system containing a specific sequence or the TRIM25-PGAM5 interaction blocking peptide, thereby cleaving mtDNA release-cGAS-STING and blocking NLRP3 inflammasome activation, thereby alleviating tissue damage and inflammatory cell death caused by ischemia-reperfusion. The ischemia-reperfusion injury mentioned above is intestinal ischemia-reperfusion injury caused by various traumas and surgical etiologies; The sequence of the TRIM25-siRNA is as follows: the sequence of the sense strand is 5'-GCUCGAUACCAUCUACCAGTT-3', and the sequence of the antisense strand is 5'-CUGGUAGAUGGUAUCGAGCTT-3'. The TRIM25-targeting inhibitor is administered orally or via intraperitoneal injection. The TRIM25-siRNA delivery system is administered orally, 24 hours before the onset of ischemic injury. The TRIM25-PGAM5 interaction blocking peptide is administered via intraperitoneal injection, at the onset of reperfusion.

[0007] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention inhibits IRI-related mitochondrial damage, inflammatory cell death, and excessive inflammation at the source by specifically blocking the TRIM25-PGAM5-mtDNA-cGAS-STING-NLRP3 signaling axis.

[0008] This invention significantly reduces histopathological damage (such as intestinal villus destruction and epithelial detachment) after ischemia-reperfusion, improves cell viability, and reduces the release of inflammatory factors (IL-1β, IL-18).

[0009] This invention is applicable to the treatment of IRI in various organs, and is particularly effective for organs with mitochondrial enrichment such as the intestines, liver, and heart, with a wide range of applications.

[0010] This invention is based on a well-defined molecular mechanism, has high safety and strong targeting, and avoids the non-specific side effects of traditional anti-inflammatory drugs. Attached Figure Description

[0011] 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 1In this embodiment of the invention, pyroptosis is involved in and exacerbates the occurrence of ischemia-reperfusion injury (I / R) and hypoxia / reoxygenation (H / R) treatment of small intestinal crypt epithelial cells (IEC-6) in mice; (A) Real-time dynamic monitoring of blood flow changes using laser speckle contrast imaging confirmed the successful establishment of a mouse intestinal ischemia / reperfusion injury model; (B) H&E staining and Chiu's score (n=6, Bars=200µm) of mouse intestinal tissue after ischemia / reperfusion injury; (C) Western blot results and quantitative analysis of HIF-1α protein expression levels in in vitro experiments; (D) Cell viability was detected using the CCK-8 assay; (E) Western blot results and quantitative analysis of NLRP3, caspase-1, and N-GSDMD protein expression levels in in vivo experiments; (F) Western blot results and quantitative analysis of NLRP3, caspase-1, and N-GSDMD protein expression levels in in vitro experiments. n =3, * indicates comparison with SHAM or Control, # indicates comparison with 90 or 120 minutes, * p <0.05,** p <0.01; # p <0.05, ## p <0.01.

[0012] Figure 2 This is a schematic diagram illustrating TRIM25 as a key regulatory factor in cellular response to intestinal ischemia-reperfusion injury in this embodiment of the invention; (A) Venn diagram showing the results of screening key genes from the GeneCards and GEO databases; (B) Western blot results and quantitative analysis of TRIM25 protein expression levels in in vivo experiments; (C) Western blot results and quantitative analysis of TRIM25 protein expression levels in in vitro experiments; (D) qRT-PCR detection and quantitative analysis of TRIM25 mRNA expression levels in in vitro and in vivo experiments; (E) Immunohistochemical fluorescence image of TRIM25 protein expression in mouse intestinal tissue (Bars=200µm); (F) Immunofluorescence image of TRIM25 protein expression in rat IEC-6 intestinal epithelial cells and the corresponding 2.5D view (Bars=10µm); n =3, * indicates comparison with SHAM or Control, * p <0.05,** p <0.01.

[0013] Figure 3In this embodiment of the invention, TRIM25 exacerbates intestinal ischemia / reperfusion injury by synergistically regulating the NLRP3 inflammasome and the cGAS-STING pathway; (A) Western blot experiment and quantitative analysis to detect changes in transfection efficiency after TRIM25 knockdown transfection of IEC-6 cells; (B) Western blot experiment and quantitative analysis to detect changes in pyroptosis markers (NLRP3, caspase-1, N-GSDMD) after TRIM25 knockdown; (C) qRT-PCR experiment and quantitative analysis to detect changes in pyroptosis markers (IL-18, IL-1β) after TRIM25 knockdown; (D) Immunofluorescence and visualization of NLRP3 and caspase-1 in IEC-6 cells (Bars=10µm); (E) Western blot analysis. (F) Immunofluorescence and 2.5D visualization of cGAS in IEC-6 cells (Bars=10µm); (G) Western blot and quantitative analysis to detect the protein expression levels of cGAS-STING pathway markers (NF-κBp65, cGAS, p-IRF3, STING) in IEC-6 cells after TRIM25 gene knockdown; (H) Immunofluorescence and 2.5D visualization of cGAS in IEC-6 cells after TRIM25 gene knockdown (Bars=10µm). n =3, * indicates comparison with Control+si-NC, # indicates comparison with H / R+si-NC, * p <0.05,** p <0.01, *** p <0.001, **** p <0.0001, # p <0.05, ## p <0.01, ### p <0.001.

[0014] Figure 4 This is a schematic diagram illustrating how cGAS deficiency can improve pyroptosis and tissue damage in I / R-treated mice or H / R-treated IEC-6 mice in the embodiments of the present invention; (AB) H&E staining and Chiu's score detection of the pre-protective effect of the cGAS-specific inhibitor RU.521 on intestinal tissue in mouse intestinal ischemia-reperfusion injury. n=6, Bars=100µm); (C) Western Blot experiment and quantitative analysis to detect the protein expression level of pyroptosis markers in cells after administration of the cGAS-specific inhibitor RU.521 in vivo; (D) Western Blot experiment and quantitative analysis to detect the protein expression level of pyroptosis markers in IEC-6 cells after administration of the cGAS-specific inhibitor RU.521; (E) qRT-PCR experiment and quantitative analysis to detect the mRNA expression level of inflammatory factors (IL-18, IL-1β) in cells after administration of the cGAS-specific inhibitor RU.521 in vitro; (F) Western Blot experiment and quantitative analysis to detect the protein expression level of pyroptosis markers in cells after knockdown of the cGAS gene in vitro; (G) qRT-PCR experiment and quantitative analysis to detect the mRNA expression level of inflammatory factors (IL-18, IL-1β) in cells after knockdown of the cGAS gene in vitro; (H) Immunofluorescence and 2.5D visualization of NLRP3 cells after knockdown of the cGAS gene in IEC-6 cells; n =3, Bars=10µm, * indicates comparison with Control+si-NC, # indicates comparison with H / R+si-NC; p <0.05,** p <0.01, *** p <0.001, # p <0.05, ## p <0.01, ### p <0.001.

[0015] Figure 5 In this embodiment of the invention, TRIM25 deficiency alleviated mitochondrial damage and mtDNA release in H / R-treated IEC-6 cells; (A) Mito SOXRed staining images showing the effect of TRIM25 gene knockout on H / R-treated or untreated mitochondrial reactive oxygen species assays (Bars=5µm); (B) JC-1 staining images showing the effect of TRIM25 gene knockout on H / R-treated or untreated mitochondrial transmembrane potential assays (Bars=5µm); (C) The effect of TRIM25 gene knockout on the enrichment of cytoplasmic mtDNA (mt-ND1, mt-ND4, and mt-Cytb) fragments; (D) Transmission electron microscopy (TEM) images showing the effect of TRIM25 gene knockout on H / R-treated or untreated mitochondrial structure (Bars=1µm). n =3, * indicates comparison with Control+si-NC, # indicates comparison with H / R+si-NC; *** p <0.001, **** p <0.0001,# p <0.05, ### p <0.001.

[0016] Figure 6 This is a schematic diagram illustrating how TRIM25 promotes the extracellular release of mitochondrial DNA via the mitochondrial outer membrane protein PGAM5 in this invention embodiment; (A) Venn diagram screening of core genes; (B) Immunoprecipitation experiment to verify the interaction between TRIM25 and PGAM5; (C) Western blotting experiment and quantitative analysis to detect changes in PGAM5 protein expression levels in vivo; (D) Western blotting experiment and quantitative analysis to detect changes in PGAM5 protein expression levels after TRIM25 knockdown or overexpression in vitro; (E) MitoSOXRed staining image showing the effect of PGAM5 gene knockdown on H / R treatment or no treatment for mitochondrial reactive oxygen species assay (Bars=5µm); (F) JC-1 staining showing the effect of PGAM5 gene knockdown on H / R treatment or no treatment for mitochondrial transmembrane potential assay (Bars=5µm); (G) Effect of PGAM5 gene knockdown on the enrichment of cytoplasmic mtDNA (mt-ND1, mt-ND4 and mt-CytB) fragments; (H) Western blotting. Blot experiments and quantitative analysis were used to detect changes in the protein expression levels of TRIM25, PGAM5, pyroptosis markers (NLRP3, caspase-1, N-GSDMD), and cGAS-STING signaling pathway markers (cGAS, STING) after TRIM25 overexpression and PGAM5 knockdown in vitro; (I) Mito SOX Red staining images showed the effect of TRIM25 overexpression and PGAM5 knockdown on H / R treatment or no treatment of mitochondrial reactive oxygen species (ROS) assay (Bars=5µm); (J) JC-1 staining showed the effect of TRIM25 overexpression and PGAM5 knockdown on H / R treatment or no treatment of mitochondrial transmembrane potential assay (Bars=5µm); (K) Effect of TRIM25 overexpression and PGAM5 knockdown on cytoplasmic mtDNA (mt-ND1, mt-ND4, and mt-CytB) fragment enrichment; (L) Western blot experiments were used to preliminarily detect the potential ubiquitination modification effect of TRIM25 and PGAM5. n =3, * indicates comparison with SHAM, Control, Control+si-NC, and Vector, # indicates comparison with H / R+si-NC and pcDNA-TRIM25+si-NC, * p <0.05,** p <0.01, *** p <0.001, #p <0.05, ## p <0.01, ### p <0.001.

[0017] Figure 7 This diagram illustrates the mechanism by which TRIM25 regulates pyroptosis in intestinal ischemia-reperfusion injury in an embodiment of the present invention. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0019] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] This invention provides a method for improving ischemia-reperfusion injury by inhibiting TRIM25, characterized in that the method includes blocking downstream mitochondrial damage, mtDNA release and inflammatory pathway activation by inhibiting the expression of TRIM25 or its interaction with PGAM5; The inhibition of TRIM25 refers to the administration of a TRIM25-targeting inhibitor, which inhibits TRIM25 expression or blocks the interaction between TRIM25 and PGAM5 by inhibiting the TRIM25-siRNA drug delivery system containing a specific sequence or the TRIM25-PGAM5 interaction blocking peptide, thereby cleaving mtDNA release-cGAS-STING and blocking NLRP3 inflammasome activation, thereby alleviating tissue damage and inflammatory cell death caused by ischemia-reperfusion. The ischemia-reperfusion injury mentioned above is intestinal ischemia-reperfusion injury caused by various traumas and surgical etiologies; The sequence of the TRIM25-siRNA is as follows: the sense strand sequence is 5'-GCUCGAUACCAUCUACCAGTT-3', and the antisense strand sequence is 5'-CUGGUAGAUGGUAUCGAGCTT-3'. The TRIM25-targeting inhibitor is administered orally or via intraperitoneal injection. The TRIM25-siRNA delivery system is administered orally, 24 hours before the onset of ischemic injury. The TRIM25-PGAM5 interaction blocking peptide is administered via intraperitoneal injection, at the onset of reperfusion.

[0024] In this invention, the term "administration" refers to the provision of a medicine or pharmaceutical compound to a subject, including but not limited to administration by a medical professional and self-administration.

[0025] The term "inhibitor" refers to a substance, agent, or molecule that causes a detectable reduction (compared to a control) in the expression or activity level of a target gene.

[0026] The specific implementation and experimental methods are as follows: 1. Materials and Methods 1.1 Animal models and intestinal I / R-induced intestinal injury models All animal experiments were approved by the Animal Ethics Committee of Dalian Medical University. Forty male C57BL / 6 mice (6-8 weeks old, weighing 18-22g) were used by the Laboratory Animal Center of Dalian Medical University (Dalian, China; License No. SCXK2020-0001). All procedures were performed in accordance with the guidelines of the National Institutes of Health (NIH). Mice were randomly assigned to five groups (n=6 per group): one sham-operated group and four ischemia-reperfusion (I / R) groups. Anesthesia was induced throughout the surgery by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After laparotomy, the superior mesenteric artery (SMA) was carefully exposed. In the sham-operated group, the abdomen was closed after only the SMA was isolated. In the I / R groups, ischemia was induced by laparotomy to expose and clamp the superior mesenteric artery (SMA) for 45 minutes, followed by reperfusion for 45, 90, 180, or 360 minutes. Laser speckle contrast imaging technology was used to monitor blood flow in real time to verify the success of the model, and no animals died during the operation.

[0027] 1.2 Cell culture and hypoxia / reoxygenation (H / R) model Rat small intestinal crypt epithelial cells (IEC-6) obtained from the American Type Culture Collection (ATCC) were cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and saturated humidity. For all experiments, cells were passaged using a 0.25% trypsin solution containing EDTA. To establish a hypoxia-reoxygenation (H / R) model, normal IEC-6 cells were placed in a hypoxic chamber set at 37°C, 5% CO2, 1% O2, and 95% N2 for 4 hours (hypoxia phase). Cells were then reoxygenated by returning them to normoxic conditions (37°C, 5% CO2, 21% O2) for 0, 120, 720, or 1440 minutes. Since cell morphology and viability were virtually identical across all control groups, a single control group was used as a representative sample throughout the study. All experiments used cells from fewer than 8 generations.

[0028] 1.3 Animal administration The cGAS-specific inhibitor RU.521 was dissolved in 10% DMSO. One hour before constructing the intestinal I / R model, mice in the experimental group were injected intraperitoneally with this solution (5 mg / kg), while the control group was injected with an equal volume of physiological saline.

[0029] 1.4 Animal histopathological examination After fixing the tissues in 4% paraformaldehyde for 24 hours, they underwent standard histological procedures, including dehydration, paraffin embedding, sectioning, dewaxing, staining, dehydration, and mounting. Pathological changes in the intestinal tissues were observed under an upright optical microscope, and the degree of intestinal damage was assessed using the Chiu's scoring system.

[0030] 1.5 Cell transfection IEC-6 cells were transfected in 6-well plates at 60–70% confluency. For plasmid transfection, a mixture of 8 μL M transfection reagent per well and 3 μg trim25 overexpression plasmid or empty vector control was prepared and applied to the cells. After 6 hours of incubation, the medium was replaced with complete medium. For siRNA transfection, 7.5 μL M transfection reagent per well was combined with 7.5 μL of siRNA targeting TRIM25, PGAM5, cGAS, or a negative control siRNA. The complex was added to the cells, incubated for 6 hours, and then replaced with antibiotic-free complete medium. Cells were harvested 48 hours post-transfection for subsequent experiments.

[0031] In this invention, the sequences required for transfection are as follows: the si-TRIM25 positive strand sequence is 5'-GCUCGAUACCAUCUACCAGTT-3', the si-TRIM25 antisense strand sequence is 5'-CUGGUAGAUGGUAUCGAGCTT-3'; the si-PGAM5 positive strand sequence is 5'-CACUGUCACUCAUUAAUUCTT-3'; the si-PGAM5 antisense strand sequence is 5'-AGGUUAAUGAGUGACAGUGTT-3'; the si-cGAS positive strand sequence is 5'-GCGGCCGAGACGGUGAAUATT-3'; and the si-cGAS antisense strand sequence is 5'-UAUUCACCGUCUCGGCCGCTT-3'.

[0032] 1.6 Cell-based drug delivery The cGAS-specific inhibitor RU.521 was prepared by dissolving the cells in 10% DMSO. Before establishing the H / R model, the experimental group cells were pretreated with medium containing 10 μM RU.521, while the control group was given blank medium containing an equal volume of solvent. Cells were incubated at 37°C in a 5% CO2 incubator for 1 hour. Drug intervention was then maintained until the end of the hypoxic phase, after which the cells were replaced with complete medium and cultured until the experimental endpoint was reached.

[0033] 1.7 Cell viability assessment Cell viability was assessed using the CCK-8 reagent kit according to the manufacturer's instructions. Cells were placed in a hypoxic environment for 4 hours, then transferred to an aerobic environment and reoxygenated at 37°C for 0, 120, 720, or 1440 minutes. After reoxygenation, 10 μL of CCK-8 reagent was added to each well containing 90 μL of culture medium. The culture plate was incubated at 37°C for 45 minutes, and the absorbance was measured at 450 nm.

[0034] 1.8 Extraction of tissue and cell proteins Extraction of tissue proteins: Intestinal tissue samples were rinsed with pre-chilled PBS buffer, blotted dry with filter paper, and 50 mg of tissue was accurately weighed and transferred to a pre-chilled centrifuge tube. 1 mL of freshly prepared pre-chilled lysis buffer (RIPA lysis buffer and PMSF protease inhibitor mixed at a volume ratio of 100:1) was added. The centrifuge tube was fixed in an ice bath, and the tissue block was thoroughly homogenized using a homogenizer tip, repeating the process until the tissue was completely broken down and no visible particles remained. The homogenate was centrifuged at 12000 g for 10 min in a 4°C centrifuge. After centrifugation, the supernatant was carefully transferred to another pre-chilled centrifuge tube; this supernatant is the extracted total protein solution.

[0035] Cell protein extraction: Discard the cell culture medium in the incubator and wash the cells with pre-chilled PBS buffer. Using a six-well plate as an example, add 150 μL of pre-chilled lysis buffer to each well, ensuring it evenly covers the cell surface. Incubate the plate on ice for 15 minutes to promote complete cell lysis. Quickly scrape the cells with a cell scraper, collecting the viscous lysis products into pre-chilled centrifuge tubes, and continue lysis at 4°C for another 15 minutes. Centrifuge at 12000g for 10 minutes at 4°C, retaining the supernatant as the total protein sample.

[0036] 1.9 BCA protein concentration determination The BCA working solution was prepared according to the kit instructions. Three replicates were created for each sample in a 96-well plate. 200 µL of working solution, 18 µL of PBS diluent, and 2 µL of sample were added to each well sequentially. A standard curve was constructed using serially diluted protein standards. The plate was incubated at 37°C in the dark for 30 minutes. The absorbance at 562 nm was then measured using a microplate reader, and the protein concentration of each sample was calculated based on the standard curve. The protein concentration of all samples was then adjusted to the same level using RIPA lysis buffer. 2× Loading buffer was added and mixed thoroughly. The mixture was heated at 100°C for 5 minutes to completely denature the protein. The final product was stored at -20°C for later use.

[0037] 1.10 Protein Immunoblotting SDS-PAGE electrophoresis was employed. The specific procedure was as follows: Separating and stacking gels of appropriate concentrations were prepared according to the protein molecular weight. After complete solidification, they were assembled into the electrophoresis apparatus, and electrophoresis buffer was injected. Pre-stained protein molecular weight standards and an equal volume of protein sample were added sequentially to the sample wells. Initially, electrophoresis was performed at a constant voltage of 80 V for approximately 50 minutes to encourage sample entry into the separating gel. The voltage was then adjusted to 120 V and electrophoresis continued for approximately 50 minutes until the bromophenol blue indicator migrated to the bottom of the gel. After electrophoresis, the target protein band was located and cut according to the molecular weight standards. This band was then combined with a methanol-pretreated PVDF membrane to construct a transfer "sandwich" structure. Wet transfer was performed under a constant current of 300 mA. After transfer, the membrane was blocked in 5% BSA solution at room temperature for 120 minutes, washed with TBST buffer, and incubated overnight at 4°C with a specific primary antibody. The next day, the primary antibody was recovered and thoroughly rinsed with TBST. HRP-labeled secondary antibody was added and incubated at room temperature for 60 minutes. After washing again, enhanced chemiluminescent substrate working solution was added. The signal intensity was captured using the AI600 imaging system and analyzed using ImageJ.

[0038] 1.11 Isolation of mitochondria from cells Cells were washed with PBS and then digested with trypsin. The digested cells were collected by centrifugation at 100g for 10 minutes. The cell pellet was then gently resuspended in pre-chilled PBS and counted. The cells were then centrifuged again at 4°C and 600g for 5 minutes, and the supernatant was discarded. 1–2.5 mL of pre-chilled mitochondrial separation reagent was added to every 20-50 million cell pellets, and the cells were gently resuspended and incubated on ice for 15 minutes. The cells were then homogenized approximately 20 times using a glass homogenizer. The homogenate was then centrifuged sequentially at 4°C and 600g for 10 minutes to collect the supernatant, followed by centrifugation at 4°C and 11000g for 10 minutes to collect the supernatant. Finally, the supernatant was centrifuged at 4°C and 12000g for 10 minutes. The resulting supernatant is the cytoplasmic protein fraction after mitochondrial removal.

[0039] 1.12 Extraction of Cellular DNA Genomic DNA was isolated from cultured cells using a genomic DNA extraction kit. The specific steps were as follows: Cells were harvested after digestion, resuspended and washed with PBS buffer, centrifuged, and the supernatant was discarded. 200 µL of GA buffer was added to the cell pellet, and the pellet was vortexed thoroughly to resuspend it. Then, 20 µL of proteinase K and 200 µL of GB buffer were added sequentially, and the mixture was thoroughly mixed and incubated in a 70°C water bath for 10 minutes until the liquid became clear. 200 µL of anhydrous ethanol was added to the lysis product and vortexed to mix. The entire mixture was transferred to a CB3 adsorption column and centrifuged at 12000 rpm for 30 seconds. The filtrate was collected and discarded. The adsorption column was washed sequentially with 500 µL of GD buffer and 600 µL of PW wash buffer, with the PW wash buffer being repeated twice. After each wash, the column was centrifuged at 12000 rpm for 30 seconds, and the filtrate was discarded. After washing, the empty adsorption column was centrifuged at 12000 rpm for 2 minutes to completely remove residual ethanol, and then left uncovered for 2-3 minutes to allow the ethanol to evaporate. 50-100 µL of preheated TE elution buffer was added along the center of the adsorption membrane, and after incubation at room temperature for 5 minutes, the column was centrifuged at 12000 rpm for 2 minutes to collect the eluted genomic DNA. The DNA concentration and purity were determined using a NanoDrop 2000 micro-spectrophotometer, and the DNA product was stored at -20°C for later use.

[0040] 1.13 RNA extraction and quantitative real-time PCR Total RNA was isolated from cells using RNA Key Reagent reagent according to the manufacturer's instructions. The concentration and purity of the extracted RNA were determined using a NanoDrop 2000 micro-volume spectrophotometer. Samples were diluted as needed to obtain the optimal template concentration for subsequent reactions. First-strand cDNA synthesis was performed using a transcriptome first-strand cDNA synthesis kit. Quantitative real-time PCR was performed on a real-time PCR system with SYBR green fluorescence detection. The relative expression levels of target genes were calculated based on fluorescence signal intensity. mRNA expression levels were normalized to β-actin, while mitochondrial gene expression was normalized to gRNA18S. Using 2- ΔΔCt The method determined the relative mRNA level (n=3).

[0041] 1.14 Tissue Immunofluorescence Assay Remove frozen sections from the -20°C freezer and allow them to equilibrate to room temperature for 30 minutes. For antigen retrieval, immerse the specimen in preheated EDTA retrieval buffer (pH 9.0) and maintain this temperature using microwave irradiation or a constant-temperature water bath for 15-20 minutes. After allowing it to cool naturally to ambient temperature, wash three times with PBS buffer, five minutes apart. To enhance cell membrane permeability, apply PBS solution containing 0.3% Triton X-100 and treat at room temperature for 20 minutes. After PBS washing, apply blocking buffer prepared with 0.3% Triton X-100 and 5% BSA and block at room temperature for 60 minutes. After removing the blocking buffer, directly add the primary antibody working solution diluted with the blocking buffer, ensuring complete tissue coverage. Place the sections in a humidified chamber and incubate overnight at 4°C. The next day, recover the primary antibody and wash three times with PBST buffer, five minutes each time. Apply fluorescently labeled secondary antibody working solution (diluted with blocking buffer) to a light-protected environment and incubate at room temperature for 1 hour. Then wash three times with PBST for five minutes each time, in the dark. Counterstain with DAPI solution for 5-10 minutes at room temperature in the dark to label cell nuclei. After final rinsing with PBS, mount using anti-fluorescence quenching mounting medium. Finally, observe and acquire images under an upright fluorescence microscope.

[0042] 1.15 Cellular Immunofluorescence Experiment Cells with good morphology and 80-90% confluence were selected, washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 15 minutes, and then permeabilized with 0.1% Triton X-100 for 10 minutes. After permeabilization, the cells were washed three more times with PBS and blocked with PBS containing 5% BSA at 37°C for 60 minutes. The cells were then incubated overnight with the corresponding primary antibody at 4°C. The next day, after washing with PBS, the cells were incubated with fluorescently labeled secondary antibody at room temperature for 1 hour. After washing again, the cell nuclei were counterstained with DAPI for 10 minutes. Finally, images were acquired using an inverted fluorescence microscope.

[0043] 1.16 Detection of Mitochondrial Reactive Oxygen Species Mitosolic superoxide dismutase (MOD) was detected using MitoSOX Red fluorescent probe staining. A monolayer of adherent cells from a six-well culture plate was taken, the old culture medium was removed, and the cells were washed twice with PBS buffer. Then, 1 mL of MitoSOX Red staining working solution was added, and the cells were incubated at 37°C in the dark for 30 minutes. After incubation, the staining solution was aspirated, and the cells were washed twice with PBS. Finally, 2 mL of DMEM culture medium was added to cover the cell layer. Visible cells were directly imaged using an inverted fluorescence microscope, and the degree of MOD generation was semi-quantitatively analyzed based on fluorescence intensity.

[0044] 1.17 Mitochondrial membrane potential detection Live cells were stained using the JC-1 fluorescent probe to detect mitochondrial membrane potential (ΔΨm) levels. Adherent cells cultured in single wells of a six-well plate were removed from their original culture medium, washed twice with PBS buffer, and then 1 mL of fresh culture medium and 1 mL of JC-1 staining working solution were added. The mixture was gently shaken to ensure homogeneity. The plate was then incubated at 37°C in a humidified incubator for 20 minutes in the dark to allow the JC-1 probe to accumulate sufficiently within the mitochondria. After incubation, the staining solution was carefully removed, and the cells were washed twice with buffer to remove non-specific binding. Then, 2 mL of fresh culture medium was added to each well. Live cells were directly imaged using an inverted fluorescence microscope. The degree of change in mitochondrial membrane potential was semi-quantitatively evaluated based on the change in the fluorescence intensity ratio of the JC-1 monomer (green fluorescence) to the polymer (red fluorescence).

[0045] 1.18 Transmission electron microscopy observation Cells were collected when confluence reached 70%, and the culture medium was discarded. Immediately after collection, the cells were fixed in situ with electron microscopy fixative for 5 minutes at room temperature in the dark. The fixed cells were gently scraped unidirectionally with a cell scraper and transferred to centrifuge tubes using a Pasteur pipette. The tubes were centrifuged at 2500 rpm for 5 minutes to form dense cell clumps. The supernatant was discarded, and the clumps were resuspended by gentle pipetting with fresh electron microscopy fixative. A second fixation was performed at room temperature in the dark for 30 minutes, followed by overnight fixation at 4°C. The samples were then washed, dehydrated using a gradient of ethanol, and embedded in epoxy resin. Ultrathin sections of the embedded samples were prepared and analyzed using transmission electron microscopy.

[0046] 1.19 Immunoprecipitation assay Immunoprecipitation was used to study protein-protein interactions. 50 µL of magnetic beads were taken, the supernatant was discarded, and the beads were washed three times with wash buffer to remove impurities. Then, 50 µL of wash buffer and 6 µL of a specific antibody (anti-TRIM25 or anti-PGAM5) were added to the magnetic beads, and the mixture was incubated on a rotary shaker for 2 hours to allow the antibody to fully bind to the magnetic beads. After incubation, the supernatant was discarded, and the antibody-coated magnetic beads were mixed with 700 µL of the target protein lysis supernatant. The mixture was incubated on a rotary shaker for another 4 hours to capture the target protein complex. After incubation, the supernatant was removed, and the magnetic beads were resuspended in 50 µL of 1×SDS loading buffer and boiled for 5 minutes to denature the protein. Finally, the supernatant was collected and stored at -20°C for later use.

[0047] 1.20 Immunoprecipitation-Mass Spectrometry Immunoprecipitation combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to identify the target protein-interacting proteins. The specific steps were as follows: Protein samples were separated by SDS-PAGE. Electrophoresis was stopped when the bromophenol blue front penetrated approximately 1 cm into the separating gel. The gel was then removed, stained with Coomassie Brilliant Blue for 1 h, and destained until the background was clear. The target band (approximately 1 cm) was then cut out. 2 To ensure clear and undegraded bands, the gel was placed in an EP tube, submerged in deionized water, and stored at 4°C. The gel was then subjected to dehydration, disulfide reduction and alkylation, and trypsin digestion to extract peptides. After desalting and concentration, the peptides were analyzed by mass spectrometry. Database retrieval software was used to analyze the raw mass spectrometry data to complete protein identification.

[0048] 1.21 Statistical Analysis Statistical analysis results are presented as mean ± standard error (mean ± SEM). All experimental data were obtained based on at least three independent repeated experiments. For comparisons between two groups, unpaired Student's t-tests were used; for comparisons involving three or more groups, one-way ANOVA was employed. Data processing and image generation were performed using GraphPad Prism 9.0 statistical software and Adobe Photoshop image processing software, respectively. The statistical significance threshold was set at a p-value less than 0.05.

[0049] 2. Results 2.1 Intestinal I / R and H / R activate pyroptosis, exacerbating in vitro and in vivo damage. An I / R model was established using the superior mesenteric artery clamping method to simulate ischemia-reperfusion in vivo. Laser speckle imaging was used to capture dynamic changes in blood flow to verify the successful establishment of the I / R injury model. Figure 1 A). Subsequently, histopathological examination was performed on intestinal tissues from mice that had undergone 45 minutes of ischemia followed by reperfusion at different times (45, 90, 180, and 360 minutes). H&E staining results and Chiu's score showed that the degree of intestinal tissue damage peaked within the 90-minute reperfusion period, with pathological features including disordered small intestinal mucosal villi structure, exposed capillaries, and collapsed or even structurally damaged epithelial apex. With prolonged reperfusion time, the degree of intestinal tissue damage gradually improved. Figure 1 B). Western blot analysis was used to detect changes in the expression of hypoxia-inducible factor HIF-1α. Compared with the control group, the protein expression level of HIF-1α remained elevated after 4 hours of simple hypoxia and 120 minutes of reoxygenation after 4 hours of hypoxia, confirming the successful establishment of the H / R model. Figure 1C). Subsequently, the viability of H / R-treated IEC-6 cells was detected using the CCK-8 assay. The results showed that cell viability was significantly inhibited after 120 minutes of reoxygenation, inducing the most severe damage. However, compared with 120 minutes of reoxygenation, reducing or prolonging the reoxygenation time improved cell viability (C). Figure 1 D). Compared with the sham-operated group or the normal control group, the expression levels of pyroptosis markers NLRP3, caspase-1, and N-GSDMD proteins were significantly increased in the I / R and H / R groups. When the reperfusion or reoxygenation duration was prolonged to more than 90 minutes in vivo or more than 120 minutes in vitro, respectively, the expression levels of these three proteins decreased. Figure 1 E, F). The above experimental results show that in mice, intestinal ischemia for 45 minutes followed by reperfusion for 90 minutes, and IEC-6 cells hypoxia for 4 hours followed by reoxygenation for 120 minutes significantly activated pyroptosis and caused the most severe intestinal tissue damage. Therefore, this time is the optimal time to establish an experimental model.

[0050] 2.2 TRIM25 is a regulator of cellular response to intestinal ischemia-reperfusion injury. In the pathological process of intestinal I / R injury, the pyroptosis pathway plays an important regulatory role. Notably, the death signals and inflammatory mediators released after mitochondrial damage further aggravate the pathological damage to intestinal tissue. Based on the joint analysis of the GeneCards database and the GEO dataset (GSE37013), this invention screened 200 genes associated with pyroptosis regulation, mitochondrial damage, and intestinal I / R injury. Through systematic exclusion of low-expression proteins, proteins that have been extensively studied in intestinal epithelial cells, and proteins with sufficient literature evidence, five potential target molecules were initially screened: TRIM21, TRIM24, TRIM25, IQGAP1, and ANXA1. Figure 2 A). Meanwhile, given the low mRNA expression levels of TRIM21, TRIM24, and ANXA1 in intestinal epithelial tissue, the dynamic expression trends of these substances in this invention are highly synchronized with the peak phase of pyroptosis damage in both the intestinal tissue I / R injury model and the cell H / R model. Figure 2 Subsequent experimental data showed that TRIM25 was significantly upregulated at both the protein and transcriptional levels.

[0051] 2.3 TRIM25 exacerbates intestinal I / R injury by synergistically regulating the NLRP3 inflammasome and cGAS-STING pathway. This invention first establishes a TRIM25 knockdown model in IEC-6 cells. Western blot validation showed that targeting siRNA can efficiently silence TRIM25 expression, laying the foundation for subsequent functional experiments. Figure 3A). Based on this, the effect of TRIM25 on NLRP3 inflammasome activation was systematically evaluated. The results showed that H / R treatment significantly upregulated the protein levels of NLRP3, caspase-1, and N-GSDMD, while TRIM25 knockdown reversed the abnormally high expression of the above-mentioned pyroptosis-related molecules. Figure 3 B); qRT-PCR further confirmed that TRIM25 knockdown inhibited H / R-induced IL-18 and IL-1β mRNA transcription (B). Figure 3 C). Immunofluorescence analysis, at the spatial localization level, confirmed that TRIM25 knockdown significantly reduced the fluorescence intensity of NLRP3 and caspase-1. Figure 3 D), highly consistent with the results of Western blotting; both Western blotting and immunofluorescence results showed that the mouse intestinal I / R model and IEC-6 cell H / R treatment could significantly induce upregulation of cGAS expression ( Figure 3 E, F). Mechanistically, TRIM25 knockdown effectively inhibited the H / R-triggered cGAS-STING cascade, specifically manifested as a synchronous decline in the levels of cGAS, STING, p-IRF3, and NF-κB p65 proteins. Figure 3 G), and the cytoplasmic distribution of cGAS is significantly reduced ( Figure 3 H).

[0052] In this invention, TRIM25 exerts a dual pro-inflammatory effect in intestinal I / R injury: it drives pyroptosis by activating the NLRP3 inflammasome and amplifies type I interferon response and NF-κB-mediated inflammatory response via the cGAS-STING pathway, with both working together to disrupt intestinal epithelial homeostasis.

[0053] 2.4 cGAS gene deletion can reduce the activation of pyroptosis induced by ischemia-reperfusion. H&E staining results ( Figure 4 A) and Chiu's rating ( Figure 4 (B) shows that ischemia-reperfusion injury to the intestinal tissue is the most severe, manifested as disorganized villus structure, exposed capillaries, and collapse or even disintegration of villus tips. Pretreatment of mice with RU.521 before ischemia-reperfusion induction reduced the degree of intestinal tissue damage. Consistent with this, Western blot analysis showed that RU.521 significantly inhibited ischemia-reperfusion-induced upregulation of NLRP3, caspase-1, and N-GSDMD proteins. Figure 4 C). Similarly, in in vitro experiments, RU.521 pretreatment also significantly reduced the protein levels of NLRP3, caspase-1, and N-GSDMD in IEC-6 cells subjected to hypoxia-reoxygenation treatment. Figure 4D). qRT-PCR analysis further demonstrated that RU.521 could significantly attenuate the hypoxia-reoxygenation-induced increase in IL-18 and IL-1β mRNA expression (D). Figure 4 E). To verify the role of cGAS from a genetic perspective, this invention uses a cGAS knockout model for hypoxia-reoxygenation treatment. cGAS deletion effectively inhibits hypoxia-reoxygenation-induced NLRP3 inflammasome activation, manifested by decreased expression of NLRP3, caspase-1, and N-GSDMD proteins. Figure 4 F). Correspondingly, cGAS knockout reversed the upregulation of IL-18 and IL-1β transcription levels (F). Figure 4 G). Furthermore, immunofluorescence analysis showed that NLRP3 signal was enhanced after hypoxia-reoxygenation treatment, while the signal was significantly weakened after cGAS knockdown. Figure 4 H).

[0054] according to Figure 4 It is evident that the cGAS-STING pathway plays a crucial role in promoting NLRP3 inflammasome activation and intestinal inflammation following ischemia-reperfusion injury.

[0055] 2.5 TRIM25 is involved in I / R-induced mitochondrial damage and mtDNA release This invention uses the MitoSOX Red fluorescent probe to assess mitochondrial reactive oxygen species (mtROS) levels and found that H / R treatment significantly enhanced the red fluorescence signal in cells, indicating a large accumulation of mtROS. This effect was significantly weakened after TRIM25 knockdown. Figure 5 A). Furthermore, fluorescence imaging showed that the ratio of JC-1 polymer (red) to monomer (green), an indicator of mitochondrial membrane depolarization, decreased after H / R damage, and TRIM25 knockdown reversed this loss of mitochondrial membrane potential. Figure 5 B). To verify whether TRIM25 affects the release of mtDNA into the cytoplasm, this invention isolated mitochondrial-free cytoplasmic components from IEC-6 cells and quantified mtDNA levels by qRT-PCR. The results showed that H / R stimulation significantly increased the enrichment of mtDNA (mt-ND1, mt-ND4, and mt-Cytb) in the cytoplasm, while TRIM25 knockdown significantly inhibited this phenomenon. Figure 5 C). Transmission electron microscopy further revealed significant mitochondrial ultrastructural defects in H / R-treated cells, including swelling, fragmentation, disruption of membrane integrity, and loss or breakage of cristae. These pathological changes were significantly improved in TRIM25 knockdown cells (C). Figure 5 D).

[0056] according to Figure 5It is evident that the upregulation of TRIM25 in this invention is closely related to H / R-induced mitochondrial damage and mtDNA release, suggesting its involvement in the activation of downstream inflammatory signaling pathways.

[0057] 2.6 TRIM25 binds to PGAM5 to regulate mitochondrial damage and mtDNA release. To further explore the regulatory mechanism of TRIM25 in mitochondrial damage, this study employed immunoprecipitation-mass spectrometry to systematically screen proteins interacting with TRIM25. By setting screening criteria (differentially expressed gene, subcellular localization, and cell growth and death) and performing cross-matching and analysis, two core candidate genes, VDAC3 and PGAM5, were ultimately identified. Based on gene expression level data from the Human Protein Atlas database, and considering that PGAM5 is an outer mitochondrial membrane protein and a central node in regulating mitochondrial homeostasis, cell death, and stress response, it was positioned as a key sensor and effector in mitochondrial damage signaling. Therefore, PGAM5, with moderate expression levels, was selected as a key candidate gene for subsequent mechanistic studies. Figure 6 A). To explore the potential direct interaction between the two, this invention confirmed through immunoprecipitation experiments that TRIM25 can directly bind to PGAM5 (A). Figure 6 B). Further Western blot analysis showed that PGAM5 expression levels were significantly elevated in the intestinal ischemia-reperfusion injury model (B). Figure 6 C), and its expression changes show a synergistic regulatory trend with the knockdown or overexpression of TRIM25 (C). Figure 6 D). This invention knocks down PGAM5 expression in IEC-6 cells. MitoSOX Red staining showed that hypoxia / reoxygenation treatment increased mitochondrial reactive oxygen species accumulation, while this phenomenon was significantly weakened in PGAM5 deficiency. Figure 6 E). JC-1 fluorescence detection showed that the mitochondrial membrane depolarization reflected by the decrease in the polymer / monomer ratio after hypoxia / reoxygenation treatment could also be reversed by PGAM5 knockdown ( Figure 6 F). Furthermore, qRT-PCR analysis of organelle-free cytoplasmic components showed that hypoxia / reoxygenation-induced enrichment of mt-ND1, mt-ND4, and mt-CytB all depended on the presence of PGAM5 (F). Figure 6 G). This invention overexpresses TRIM25 while simultaneously knocking down PGAM5 under H / R conditions. Western blot results showed that TRIM25 overexpression significantly increased the protein levels of NLRP3, cGAS, caspase-1, N-GSDMD, and STING, and these effects were all reversed by PGAM5 knockdown. Figure 6H). Immunofluorescence staining confirmed that PGAM5 knockdown could alleviate TRIM25-induced mtROS accumulation (H). Figure 6 I) and loss of mitochondrial membrane potential ( Figure 6 J). Correspondingly, qRT-PCR analysis showed that PGAM5 deficiency inhibited TRIM25-promoted cytoplasmic mtDNA release (J). Figure 6 K). This hypothesis was subsequently verified through ubiquitination experiments, which showed that knocking down TRIM25 reduced the ubiquitination level of PGAM5 (K). Figure 6 L) according to Figure 6 It is evident that this invention demonstrates that TRIM25 initiates mtDNA release, activates the cGAS-STING pathway, and promotes NLRP3 inflammasome assembly by directly interacting with PGAM5, in which PGAM5 is a key mediator of TRIM25-driven mitochondrial dysfunction and inflammatory response.

[0058] Combination Figure 1-6 This invention elucidates a novel inflammatory pathway in intestinal ischemia / reperfusion injury centered on the TRIM25–PGAM5 mitochondrial signaling axis. TRIM25 is upregulated after ischemia / reperfusion and promotes NLRP3 inflammasome-mediated pyroptosis. TRIM25 directly interacts with PGAM5, increasing mitochondrial membrane permeability and promoting the release of mtDNA into the cytoplasm. The released mtDNA is recognized by cGAS, thereby activating STING and initiating an NLRP3-dependent pyroptosis program. Figure 7 ).

[0059] In summary, TRIM25 senses ischemic stress and, in synergy with PGAM5, disrupts mitochondrial integrity, activates the cGAS–STING–NLRP3 axis, and ultimately drives inflammatory cell death. Correspondingly, this invention inhibits the expression of TRIM25 or the interaction of the TRIM25-PGAM5 signaling axis, thereby inhibiting mtDNA-cGAS-STING signal transduction and NLRP3-dependent pyroptosis, blocking downstream mitochondrial damage, mtDNA release, and inflammatory pathway activation, which can effectively improve ischemia-reperfusion injury.

[0060] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is determined by the appended claims.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for improving ischemia-reperfusion injury by inhibiting TRIM25, characterized in that, The method includes blocking downstream mitochondrial damage, mtDNA release, and activation of inflammatory pathways by inhibiting the expression of TRIM25 or its interaction with PGAM5. The inhibition of TRIM25 refers to the administration of a TRIM25-targeting inhibitor, which inhibits TRIM25 expression or blocks the interaction between TRIM25 and PGAM5 by inhibiting the TRIM25-siRNA drug delivery system containing a specific sequence or the TRIM25-PGAM5 interaction blocking peptide, thereby cleaving mtDNA release-cGAS-STING and blocking NLRP3 inflammasome activation, thereby alleviating tissue damage and inflammatory cell death caused by ischemia-reperfusion. The ischemia-reperfusion injury mentioned above is intestinal ischemia-reperfusion injury caused by various traumas and surgical etiologies; The sequence of the TRIM25-siRNA is as follows: the sense strand sequence is 5'-GCUCGAUACCAUCUACCAGTT-3', and the antisense strand sequence is 5'-CUGGUAGAUGGUAUCGAGCTT-3'. The TRIM25-targeting inhibitor is administered orally or via intraperitoneal injection. The TRIM25-siRNA delivery system is administered orally, 24 hours before the onset of ischemic injury. The TRIM25-PGAM5 interaction blocking peptide is administered via intraperitoneal injection, at the onset of reperfusion.

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