siRNA targeting interleukin-24 gene and its use in treating sepsis

Abstract

The application discloses siRNA targeting interleukin-24 genes and application thereof in treatment of sepsis, and belongs to the technical field of biological medicine. The nucleotide sequence of the siRNA comprises a sense strand shown in SEQ ID NO. 1 and an antisense strand shown in SEQ ID NO. 2; the 5' end of the sense strand is modified by cholesterol, and the 3' end of the antisense strand is modified by methoxy. It is found through animal experiments that the siRNA can effectively inhibit excessive inflammatory response in a sepsis model and significantly improve the survival rate, thereby providing a brand-new candidate drug and a precise target for treatment of sepsis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to siRNA targeting the interleukin-24 gene and its application in the treatment of sepsis. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. It has extremely high global morbidity and mortality rates, placing a heavy burden on healthcare systems. Currently, there is a lack of specific treatments targeting the core pathology of sepsis, with treatment primarily relying on antibiotics and organ support, but long-term prognosis for patients is poor. Early sepsis is characterized by an excessive systemic inflammatory response, namely a "cytokine storm." However, targeted therapies against classic pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) have not achieved ideal results in clinical trials, suggesting the existence of other key, under-recognized pro-inflammatory mediators driving the progression of sepsis.

[0003] Interleukin-24 (IL-24) is a member of the IL-10 cytokine family and is expressed in a variety of immune cells. Current research indicates that IL-24 exhibits pro-inflammatory properties in autoimmune diseases and certain infection models, inducing the secretion of other pro-inflammatory factors (such as IL-6 and TNF-α) and activating immune cells. However, the specific role, mechanism, and therapeutic potential of IL-24 in sepsis, a disease characterized by uncontrolled systemic inflammation, were previously completely unknown.

[0004] Small interfering RNA (siRNA) technology can specifically silence the expression of target genes at the post-transcriptional level through RNA interference (RNAi) mechanisms, providing a new strategy for targeting traditionally "undruggable" cytokines. Previous studies have explored using siRNA to target other targets in sepsis; for example, using siRNA targeting eIF-5A to downregulate pro-inflammatory cytokines for sepsis treatment, and loading CCR2-targeting siRNA (siCCR2) into macrophage-derived exosomes to reduce septic cytokine storms. Furthermore, siRNAs targeting sepsis-related targets such as GSDMD are in preclinical development. These works demonstrate the feasibility of siRNA therapy in the treatment of sepsis. However, there are currently no publicly reported specific siRNAs targeting IL-24, a newly discovered key pro-inflammatory target, and their therapeutic applications. Summary of the Invention

[0005] The purpose of this invention is to provide siRNA targeting the interleukin-24 gene and its application in the treatment of sepsis, in order to solve the problems existing in the prior art. It reveals and verifies for the first time that IL-24 is a key pro-inflammatory factor in the development of sepsis, and based on this discovery, an siRNA that can efficiently silence IL-24 expression was designed. This siRNA can effectively inhibit the excessive inflammatory response in a sepsis model and significantly improve the survival rate, providing a novel candidate drug and precise target for the treatment of sepsis.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides siRNA targeting the interleukin-24 gene, wherein the nucleotide sequence of the siRNA includes a sense strand as shown in SEQ ID NO.1 and an antisense strand as shown in SEQ ID NO.2.

[0007] Furthermore, in addition to the sense and antisense strands of the specific sequences mentioned above, it can also be a sequence that has at least 90% identity with the nucleotide sequence of the sense or antisense strand, more preferably at least 95% identity, and most preferably 100% identity.

[0008] Preferably, the 5' end of the sense chain is modified with cholesterol, and the 3' end of the antisense chain is modified with methoxy groups.

[0009] The present invention also provides the use of the siRNA in the preparation of medicaments for the prevention and / or treatment of sepsis.

[0010] The present invention also provides the use of the siRNA in the preparation of drugs that improve the prognosis of sepsis.

[0011] The present invention also provides the use of the siRNA in the preparation of drugs that improve the survival rate of patients with sepsis.

[0012] The present invention also provides a medicament for the prevention and / or treatment of sepsis, comprising the siRNA and a pharmaceutically acceptable carrier.

[0013] The above-mentioned drugs can be administered via intravenous injection, intraperitoneal injection, subcutaneous injection, or mucosal administration. To improve delivery efficiency, the siRNA can also be encapsulated or conjugated to a delivery vector, which includes, but is not limited to, lipid nanoparticles (LNPs), exosomes (such as macrophage-derived exosomes), polymer nanoparticles, cationic liposomes, or viral vectors.

[0014] The present invention discloses the following technical effects: (1) Target innovation: This invention establishes IL-24 as a key new target for the treatment of sepsis for the first time. Through bioinformatics analysis, gene knockout animal models and gain / loss of function experiments, the core pro-inflammatory role of IL-24 in sepsis has been fully demonstrated, breaking through the bottleneck of poor efficacy of traditional cytokine targets.

[0015] (2) Highly efficient and specific molecular design: The siRNA sequence provided by this invention is rationally designed to target the effective site of IL-24 mRNA. Through advanced structural design and two chemical modification strategies, its gene silencing efficiency, nuclease stability, targeting specificity and in vivo delivery efficiency are greatly improved, while reducing off-target risk and immune stimulation side effects.

[0016] (3) Clear treatment mechanism: This invention not only provides an effective therapeutic molecule, but also elucidates its mechanism of action: that is, by inhibiting IL-24, it simultaneously weakens the two key links of sepsis exacerbation, pathogen chemotaxis and excessive recruitment of immune cells, thereby alleviating the "cytokine storm" from the source and has the potential for multi-effect treatment.

[0017] (4) Broad therapeutic prospects: Preclinical experiments have shown that intervention using the IL-24 siRNA designed in this invention can significantly improve the survival rate of sepsis model animals. This lays a solid theoretical and technical foundation for the development of a novel, nucleic acid interference-based precision treatment for sepsis. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram illustrating the principle that inhibiting IL-24 expression can slow the progression of sepsis in this invention. Figure 2 The results are from bioinformatics analysis; A and F show the expression of interleukins IL-24, IL-6, IL-1β, IL-17, and IL-1α in the blood of mice with peritonitis-induced sepsis and patients, respectively; BE shows the correlation between IL-24 expression and IL-6, IL-1β, IL-1α, and IL-17 in the blood of sepsis-affected mice, respectively; G and J show the correlation between IL-24 expression and IL-6, IL-1β, IL-1α, and IL-17 in the blood of sepsis-affected patients, respectively. Figure 3The results are as follows: A is a schematic diagram of the construction of a mouse sepsis model; B is the expression level of IL-24 in the plasma of sepsis mice; CD shows the construction and identification results of IL-24 gene knockout (IL-24 KO) mice, lanes 1-3 show the gene expression results of the IL-24 KO sepsis mouse model, lanes 4-5 show the gene expression results of the wild-type mouse model, and M is the standard DNA marker; EF shows the flowcharts and survival statistics of E. coli infection in the IL-24 KO sepsis mouse model and wild-type mice. Figure 4 In vivo experimental results to explore the mechanism; A is the evaluation of IL-24 after sepsis induction. - / - Flowcharts for bacterial load and immune cell profile analysis in WT mice; B represents spleen bacterial load; C represents liver bacterial load; D represents peritoneal neutrophil count; E represents T cell count; F represents peripheral blood leukocyte count. Figure 5 A shows the results of the in vitro mechanism verification experiment; B shows the capillary experiment flowchart; C shows the statistical results of the capillary experiment; D shows the Transwell experiment diagram; E shows the neutrophil migration; and E shows the T cell migration. Figure 6 A shows the in vivo validation of the pathogenic effect of IL-24 and the results of the investigation on its therapeutic targeting potential; B shows the experimental flowchart for confirming the pathogenic effect of IL-24; C shows the bacterial load in the spleen; D shows the statistical results of the number of white blood cells, neutrophils and T cells; E shows the experimental flowchart for evaluating the therapeutic potential of inhibiting IL-24; and F shows the statistical results of mouse survival rate. Detailed Implementation

[0020] 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.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to 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. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] This invention reveals that IL-24 is significantly overexpressed in patients with sepsis and animal models. It exacerbates early-stage excessive inflammatory responses, leading to cytokine storms and organ damage, by simultaneously promoting the chemotaxis of pathogens (such as bacteria) and immune cells (such as neutrophils and T lymphocytes) to the site of infection. Silencing IL-24 expression can break this vicious cycle, reduce systemic inflammation levels, decrease organ bacterial load, thereby improving prognosis and significantly increasing survival rates. (See technical schematic diagram below.) Figure 1 The specific techniques of the present invention will be further analyzed and described below by way of embodiments.

[0026] Example 1: Differential Gene Expression and Correlation Analysis RNA sequencing data were obtained from the Gene Expression Overall (GEO) database (GSE90727 and GSE137340). Data preprocessing was performed using the R package featureCounts. Differential expression of IL-6, IL-1β, IL-17, IL-24, and IL-1α was analyzed using DESeq2 and visualized using pheatmap. Pearson correlation coefficients between IL-24 and IL-6, IL-1β, IL-17, or IL-1α were calculated using the R language package stats.

[0027] like Figure 2 As shown, analysis of the GEO datasets (accession numbers: GSE90727 and GSE137340) revealed that in peritonitis-induced sepsis mice ( Figure 2 (A) and patients ( Figure 2In the blood of patients with inflammatory disorders (IFN-γ), inflammatory mediators, including interleukins IL-24, IL-6, IL-1β, IL-17, and IL-1α, were significantly upregulated. Correlation analysis in mice and humans showed similar results: IL-24 expression was not significantly correlated with IL-1β expression. Figure 2 C, H), and IL-6 (Pearson r = 0.4612, p<0.0079, Figure 2 B; Pearson r = 0.4888, p = 0.0007, Figure 2 Middle G), IL-1α (r = 0.8239, p<0.0001, Figure 2 D; Pearson r = 0.5598, p<0.0001, Figure 2 in I) and IL-17 (r = 0.6165, p = 0.002; Figure 2 E; Pearson r = 0.3151, p = 0.0350, Figure 2 The positive correlation between IL-24 and J indicates that IL-24 may play a role as a pro-inflammatory mediator in the cytokine network during the pathogenesis of sepsis.

[0028] Example 2: Design, synthesis, and chemical modification of siRNA targeting the human IL-24 gene 1. Target sequence selection The mRNA sequence of human IL-24 (NCBI Gene ID: 11009) (reference sequence such as NM_006850) was screened using rational design principles. Priority was given to screening 19-25 nt target sequences that met the following criteria: GC content between 30% and 60%; avoidance of long fragments (e.g., >4 single-base repetitions); and the 3' end of the antisense strand preferably being A, T, or U, and the 5' end preferably being G or C. Finally, a highly effective silencing target located in the coding region was selected.

[0029] 2. siRNA sequence Based on the selected target, the following double-stranded siRNA core sequence was designed and synthesized: Chain of Justice: 5'-CCGCAGAGCAUUCAAACAGUUTT-3' (SEQ ID NO.1); Antisense chain: 5'-AACUGUUUGAAUGCUCUGCGGTT-3' (SEQ ID NO.2).

[0030] The core sequence described above was subjected to two chemical modifications to prepare stabilized siRNA, specifically: (siRNA-IL-24-Stab): the 5' end of the sense strand of the siRNA was modified with cholesterol to promote its crossing of the cell membrane; the 3' end of the antisense strand was modified with a methoxy group to enhance its stability in vivo and prolong its half-life. All synthesis and modification work was commissioned to Qingke Biotechnology Co., Ltd.

[0031] Example 3: Therapeutic effect of IL-24 siRNA in a mouse model of sepsis 1. Laboratory animals and cells All animal experimental procedures were approved by the Laboratory Animal Ethics Committee of Guangzhou Lingfu Top Biotechnology Co., Ltd. (Approval No.: LFTOP-IACUC-2024-0058) and followed the ARRIVE guidelines. Experiments were conducted at the Specific Pathogen-Free (SPF) Laboratory Animal Center of Guangzhou Lingfu Top Biotechnology Co., Ltd. C57BL / 6 mice (6-8 weeks old) were purchased from Best Biotechnology Co., Ltd. (Guangzhou, China). C57BL / 6 IL-24 - / - Knockout (KO) mice (#S-KO-15440) were constructed using the CRISPR / Cas9 system and purchased from Cyagen Biosciences (China). Cas9 mRNA and single-stranded guide RNA (sgRNA) were introduced via high-throughput electroporation of fertilized eggs to generate IL-24 heterozygotes (IL-24). + / - Mice. By targeting IL-24 + / - PCR genotyping of offspring to identify homozygous IL-24 - / - Mice (#D7283S, Beyotime, China). All animals were acclimatized to the environment for one week before model establishment and were anesthetized by intraperitoneal injection of 20 μL of tribromoethanol 14 per gram of body weight (#M2910, Aibei Biotechnology, Nanjing, China) and then sacrificed by cervical dislocation.

[0032] Escherichia coli culture: Escherichia coli strain ATCC 25922 was kindly provided by Professor Menghua Xiong of South China University of Technology (Guangzhou, China). The bacteria were cultured in brain heart extract broth (BHI) (#HB8297-5, Hopebio, China) at 37°C. After 12 h of culture, the culture was centrifuged at 1,000 × g for 10 min at 25°C, and the precipitate was resuspended in phosphate-buffered saline (PBS; #P1020, Solarbio, China) for subsequent experiments.

[0033] 2. Escherichia coli-induced mouse sepsis model Wild-type (WT) C57BL / 6 male mice (6-8 weeks old; n = 4) were administered 5 × 10⁻⁶ mmol / L via intraperitoneal injection (ip). 7One *E. coli* bacillus was dissolved in 0.2 mL PBS. Control mice (n = 4) received 0.2 mL PBS via intraperitoneal injection. Blood was collected 4 h post-injection for serum IL-24 analysis.

[0034] 3. Experimental Methods 3.1 Group Experiment To assess survival and downstream effects, WT (n = 13) and IL-24 were administered. - / - Knockout mice (n = 12) were intraperitoneally injected with 5 × 10 7 One E. coli was dissolved in 0.2 mL PBS. After 4 h, 8 mice from each group were sacrificed for further analysis. The survival of the remaining mice was monitored at 6, 12, 18, 24, 48 and 72 hours.

[0035] To investigate the effect of recombinant IL-24 protein (rIL-24) on sepsis, C57BL / 6 mice were randomly divided into a control group (PBS, intraperitoneal injection) and an rIL-24 treatment group (1 μg, intraperitoneal injection) (n = 6 in each group). One hour after injection, all mice developed 5 × 10⁵ intraperitoneal infections. 7 One E. coli bacterium was found. The mice were sacrificed 4 hours later for further analysis.

[0036] For the gene knockdown experiment, mice were divided into two groups: small interfering RNA (siRNA) targeting IL-24 (sense strand: 5'-CCGCAGAGCAUUCAAACAGUUTT-3', SEQ ID NO.1; antisense strand: 5'-AACUGUUUGAAUGCUCUGCGGTT-3', SEQ ID NO.2; n = 14) and a negative control (NC; sense strand: 5'-UUCUCCGAACGUGUCAC-3', SEQ ID NO.3; antisense strand: 5'-ACGUGACACGUUCGGAGAATT-3', SEQ ID NO.4; n = 13). The complex was prepared according to the manufacturer's instructions using transfection reagent (#V20207005, Golden Transfer, China) and administered intraperitoneally (3.3 nmol per mouse). After 12 h, all mice were intraperitoneally infected with 5 × 10⁶ siRNAs. 7 The survival of Escherichia coli was observed at 8, 16, 24, 48 and 72 h.

[0037] 3.2 Peritoneal lavage and flow cytometry Mice were anesthetized by inhalation of isoflurane. A total of 5 mL of sterile PBS was injected intraperitoneally, and peritoneal lavage fluid was collected through an abdominal incision. Cells were precipitated by centrifugation, resuspended in FACS buffer, and stained at 4°C for 20 min with APC-conjugated anti-mouse Gr-1 antibody (#108411, BioLegend, USA) and FITC-conjugated anti-mouse CD3 antibody (#100203, BioLegend, USA). 10,000 events per sample were acquired using a DxFLEX flow cytometer (Beckman Coulter, USA), and analyzed using FlowJo software (v10.8.1, BD Biosciences, USA).

[0038] 3.3 Bacterial chemotactic capillary assay One end of a capillary tube (#5-000-2005, Drummond Scientific, USA) was filled with PBS or 10 μg / mL recombinant IL-24 (rIL-24; #HY-P700200AF, MCE, USA) / recombinant IL-19 (rIL-19; #HY-P7213, MCE) dissolved in PBS, and the other end was sealed with paraffin. The open end was immersed in 200 μL of 1×10⁻⁶ PBS. 5 Incubate the CFU / mL *E. coli* culture at 37°C for 1 h. After incubation, wash the capillary tube and transfer its contents into a microcentrifuge tube containing 50 μL PBS. Spread the sample onto BHI agar plates using an L-shaped spreader and incubate at 37°C for 12 h. Photographs and quantification of colonies are performed using a Scan® 500 colony counter (Interscience, France).

[0039] 3.4 Blood Collection Mice were anesthetized by inhalation of isoflurane, and blood was collected via the retroorbital venous plexus using EDTA-coated tubes (#CSBHC-CXG-KN-5ML, Prosperich). Blood samples from the experimental group were analyzed using a BC-5000Vet hematology analyzer (Mindray Animal Medical Technology Co., Ltd., Shenzhen, China) to determine white blood cell, neutrophil, and lymphocyte counts. Blood samples from healthy male C57BL / 6 mice (8 weeks old) were used to isolate neutrophils and CD3+. + T cells.

[0040] 3.5 Bacterial load determination Spleen and liver were collected, weighed, and homogenized in 1 mL PBS. Serial dilutions were spread onto BHI agar plates and incubated at 37°C for 12 h. Colony forming units were counted using a Scan® 500 automated colony counter (Interscience, France).

[0041] 3.6 Enzyme-linked immunosorbent assay (ELISA) Within 30 min of blood collection, the serum was centrifuged at 1,000 × g for 10 min at 25°C, collected, and diluted 10-fold with PBS. IL-24 levels were quantified (1:10 dilution) using a sandwich ELISA kit (#YNL-20180, Chengdu Yuannuo Tiancheng Technology Co., Ltd., China) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a BioTek Synergy H1 multi-plate reader (Agilent Technologies, USA), with standard concentrations ranging from 6.25 to 200 pg / mL.

[0042] 3.7 Neutrophils and CD3 + T cell isolation Neutrophils were isolated from whole blood using density gradient centrifugation (#P9201, Solarbio, China). Whole blood was diluted 1:1 with PBS, carefully added to the supernatant, and centrifuged at 1,500 × g for 30 min at 25°C. Cells were aspirated from the corresponding interfacial layers of neutrophils and lymphocytes. Red blood cells were removed from the neutrophil fraction by hypotonic lysis. Neutrophils were washed three times with PBS (500 × g, 5 min) and resuspended immediately in RPMI-1640 medium (#11875093, Gibco, USA) containing 10% fetal bovine serum (FBS, #FB25015, Clark, USA) for immediate use or stored in liquid nitrogen.

[0043] Lymphocyte viability (>95%) was confirmed by trypan blue exclusion assay (#3006793, Thermo Fisher Scientific, USA). CD3 cells were isolated using the MojoSort™ Mouse CD3 T Cell Isolation Kit (#480023, BioLegend, USA). + T cells. In short, add 100 μL of 10... 7 A suspension of cells / mL was incubated with 10 μL of antibody mixture on ice for 15 min, followed by incubation for another 15 min with 10 μL of streptavidin nanobeads. After adding 2.5 mL of buffer, cells were magnetically separated twice. The isolated CD3... + T cells were centrifuged and resuspended in RPMI-1640 containing 10% FBS for immediate experimental use or cryopreservation.

[0044] 3.8 Transwell migration experiment To assess the chemotactic effect of IL-24, neutrophils or CD3+ cells were subjected to... + T cells, including neutrophils and CD3+ cells.+ T cells were seeded in the upper chamber of a Transwell chamber (#TCS003024, Biofil, China). Standard culture medium was added to the lower chamber of the control wells, while culture medium supplemented with 1 μg / mL recombinant IL-24 was added to the lower chamber of the experimental wells. After 24 hours of incubation, cells that migrated to the lower chamber (including neutrophils and CD3+ cells) were analyzed. + T cells were observed and counted under a microscope.

[0045] 4. Results and Analysis 4.1 IL-24 is highly expressed in septic mice, and its deficiency can improve survival rate. A peritonitis-induced sepsis model was established by intraperitoneal injection of Escherichia coli. Figure 3 (A) Four hours after sepsis induction, the bacterial colony counts in mouse organs such as the spleen and liver were significantly increased, and the number of inflammatory cells in peritoneal fluid and blood was significantly increased. Therefore, four hours was determined to be the critical time point for basic modeling success. In each experiment, four hours later, mice exhibited lethargy, slow movement, curling up and immobility, and shivering or chills. Low temperature upon touching the back of the ear or paws indicated successful modeling.

[0046] ELISA results showed that, compared with the healthy control group, the serum IL-24 level in septic mice was significantly increased (p = 0.0013); Figure 3 (B) IL-24 was constructed. - / - KO mice ( Figure 3 (C) and verified through genotyping ( Figure 3 (D): From mice 1-2 weeks old, toe tissue was collected for DNA extraction and purification using a DNA extraction kit, followed by PCR amplification (IL-24). - / - For mice, the forward primer was 5'-AAAAGCACAAAGCGAGGTCAGCA-3', SEQ ID NO. 5; the reverse primer was 5'-TCCAATCAGCAGTCAGAGATGGTA-3', SEQ ID NO. 6. For wild-type mice, the forward primer was 5'-TTTACAGGTCCCCGATGTCAGAATT-3', SEQ ID NO. 7; the reverse primer chain was 5'-TCCAATCAGCAGTCAGAGATGGTA-3', SEQ ID NO. 8. Finally, agarose gel electrophoresis was performed.

[0047] Using WT and IL-24 - / - In a mouse model of sepsis ( Figure 3 (E), does not express IL-24 - / - The survival rate of KO mice was significantly higher than that of WT mice (p = 0.0046); Figure 3The results (F) indicate that IL-24 gene knockout can reduce sepsis-related mortality.

[0048] 4.2 IL-24 deficiency can reduce bacterial load and leukocyte infiltration. IL-24 was assessed 4 h after sepsis induction. - / - Bacterial load and immune cell profile in WT mice ( Figure 4 (A). Bacterial load assays showed that, compared to WT mice, IL-24... - / - The bacterial load in the spleen and liver of mice decreased by 24% and 44%, respectively (p<0.0001); Figure 4 The presence of IL-24 in peritoneal lavage fluid (BC) indicates that IL-24 promotes bacterial dissemination. Flow cytometry analysis of peritoneal lavage fluid showed that IL-24... - / - Gr1 in mice + Neutrophils (p = 0.0031) and CD3 + T cells (p = 0.0016) were significantly reduced ( Figure 4 The presence of DE (in the middle) indicates weakened recruitment of local immune cells. Furthermore, systemic analysis showed that IL-24... - / - The total white blood cell count (p = 0.0209), neutrophil count (p = 0.0412), and lymphocyte count (p = 0.0017) in mice were decreased. Figure 4 The presence of F in the middle of the spectrum indicates a weakened systemic inflammatory response.

[0049] 4.3 IL-24 enhances bacterial aggregation and immune cell chemotaxis in vitro Based on in vivo experimental results, it is hypothesized that IL-24 may exacerbate excessive inflammatory responses by promoting bacterial aggregation and immune cell chemotaxis. (Capillary assay) Figure 5 (A) indicates that, under protein gradient assay conditions, rIL-24 significantly enhanced the aggregation of Escherichia coli compared with the control group (p = 0.0079). Figure 5 (B) Transwell experiment ( Figure 5 Further analysis (C) confirmed that rIL-24 significantly increased neutrophils (p = 0.0248). Figure 5 D) and T cells (p = 0.0015; Figure 5 (E) migration to the lower chamber. These findings suggest that IL-24 exacerbates early inflammation by promoting the recruitment of bacteria and immune cells.

[0050] 4.4 In vivo validation of the pathogenic effects of IL-24 and its therapeutic targeting potential To confirm the pathogenic role of IL-24, C57BL / 6 mice were intravenously injected with rIL-24 1 hour before E. coli-induced sepsis. Figure 6(A). Four hours after injection, compared with the control group, mice treated with rIL-24 showed significantly higher bacterial loads in the spleen (p = 0.0124) and liver (p = 0.0002). Figure 6 (Bronchoscopy). Complete blood count (BC). Figure 6 The results showed that the rIL-24 group had a significant increase in white blood cells (p = 0.0009), neutrophils (p = 0.0022), and T cells (p = 0.0136).

[0051] To evaluate the therapeutic potential of inhibiting IL-24, this invention designed IL-24 siRNA with a 3' methoxy group modified to increase stability and a 5' cholesterol moiety modified to enhance cellular uptake. Twelve hours before E. coli-induced sepsis, IL-24 siRNA or a negative control was administered via intraperitoneal injection. Figure 6 (E). Mice receiving IL-24 siRNA had a 72-hour survival rate of 95%, compared to 50% in the control group (log-rank test p = 0.0136). Figure 6 The results (F) indicate that targeting and silencing IL-24 can bring significant survival benefits.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A siRNA targeting the interleukin-24 gene, characterized in that, The nucleotide sequence of the siRNA includes the sense strand as shown in SEQ ID NO.1 and the antisense strand as shown in SEQ ID NO.

2.

2. The siRNA as described in claim 1, characterized in that, The 5' end of the sense strand is modified with cholesterol, and the 3' end of the antisense strand is modified with methoxy groups.

3. The use of the siRNA as described in claim 1 or 2 in the preparation of medicaments for the prevention and / or treatment of sepsis.

4. The use of the siRNA as described in claim 1 or 2 in the preparation of drugs to improve the prognosis of sepsis.

5. The use of the siRNA as described in claim 1 or 2 in the preparation of a medicament for improving the survival rate of patients with sepsis.

6. A drug for the prevention and / or treatment of sepsis, characterized in that, Contains the siRNA as described in claim 1 or 2 and a pharmaceutically acceptable vector.

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