Use of macrophage s100a9 protein in treatment of liver fibrosis

Abstract

The present application relates to the application of targeting macrophage S100A9 protein in the treatment of liver fibrosis. The use of the composition in the preparation of a medicament for treating liver fibrosis is provided. Specifically, the composition of the present application comprises S100A9 antisense oligonucleotide, and a drug delivery carrier specifically targeting macrophages, which can load the S100A9 antisense oligonucleotide. The composition of the present application precisely targets liver macrophages, silences S100A9 protein expression, can effectively reverse liver fibrosis, and the effect is significantly better than that of existing S100A9 small molecule inhibitors, providing a reliable theoretical basis and potential technical means for clinical liver fibrosis treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of the composition in the preparation of a medicament for treating liver fibrosis. Background Technology

[0002] Liver fibrosis is a common stage in the development of chronic liver diseases caused by various etiologies. If left uncontrolled, it can worsen, progressing to cirrhosis or liver cancer, seriously threatening human health. A recent study of 5.7 million adults found prevalence rates of 44.39% for steatosis, 10.57% for severe steatosis, 2.85% for advanced liver fibrosis, and 0.87% for cirrhosis, revealing the enormous burden of liver disease we currently face.

[0003] Clinical treatment strategies for liver fibrosis are limited, currently relying mainly on treating the underlying cause, but this is only beneficial to some patients. Especially in the cirrhosis stage, clinical treatment primarily focuses on suppressing complications, which cannot achieve the goal of curing cirrhosis.

[0004] To date, research on drugs for the treatment of liver fibrosis is limited. Only retemetil, a selective thyroid hormone receptor β agonist, was approved by the U.S. Food and Drug Administration in 2024, becoming the world's first drug for treating liver fibrosis. Based on phase 3 clinical data, retemetil has shown some improvement and therapeutic effects in patients with moderate to severe liver fibrosis (F2 to F3 stages), but it also has side effects such as diarrhea, nausea, and dizziness. Furthermore, whether it can truly reduce the progression to cirrhosis and liver failure is still under phase 3 clinical investigation.

[0005] Therefore, identifying key targets in the development and progression of liver fibrosis and developing safe, effective treatments with minimal side effects that target the early stages of liver fibrosis is an important issue in the field of liver fibrosis treatment. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to provide a safe and effective new target and treatment method for liver fibrosis.

[0007] S100A9 belongs to the S100 calcium-binding protein family and is a model molecule associated with endogenous damage, playing an important role in inflammation and tissue injury. Some reports suggest that S100A9 plays a role in fibrosis in certain organs (such as the kidneys, heart, and lungs) due to its molecular mechanism.

[0008] Regarding liver fibrosis, it has been reported that S100A8 / A9 secreted by neutrophils is significantly increased in fatty liver disease-related fibrosis; the expression of S100A9 in liver fibrotic tissue is significantly increased and positively correlated with the degree of liver injury and fibrosis; these reports all suggest that inhibiting the expression of S100A9 may have potential therapeutic potential for liver fibrosis.

[0009] However, the inventors unexpectedly discovered during their research that the small-molecule inhibitor pacquimod of S100A9 did not alleviate the progression of liver fibrosis. Further investigation revealed that knocking out S100A9 in myeloid cells inhibited liver fibrosis, and this alleviating effect was independent of neutrophils that highly expressed S100A9; conversely, specific targeting and inhibition of S100A9 in macrophages effectively treated liver fibrosis. This was surprising, as previous studies have shown that S100A9 is constitutively expressed primarily in neutrophils (comprising 40% of their cytoplasmic protein) and monocytes (comprising 5% of their cytoplasmic protein) (Edgeworth J, Gorman M, Bennett R, et al Identification of p8, 14 as a highly abundant heterodimeric calcium-binding protein complex of myeloid cells. J Biol Chem, 1991); Macrophages are not the primary distribution target of S100A9, a point also confirmed in the embodiments of the present invention. Accordingly, the inventors completed the present invention.

[0010] Specifically, the technical solution adopted by the present invention to solve its technical problem is as follows.

[0011] The present invention provides the use of a composition in the preparation of a medicament for the treatment of liver fibrosis, the composition comprising an S100A9 antisense oligonucleotide and a drug delivery carrier specifically targeting macrophages, the drug delivery carrier being capable of loading the S100A9 antisense oligonucleotide.

[0012] In some embodiments, the S100A9 antisense oligonucleotide is selected from sequences shown as SEQ ID NO:2 and as shown as SEQ ID NO:3.

[0013] In some implementations, the S100A9 antisense oligonucleotide is a sequence as shown in SEQ ID NO:2.

[0014] In some implementations, the drug delivery vehicle targets macrophages through the interaction of a ligand and a receptor.

[0015] In some implementations, the drug delivery vehicle targets one or more receptors on macrophages selected from the group consisting of scavenger receptors, Toll-like receptors, integrin receptors, and C-type lectin receptors.

[0016] In some implementations, the scavenger receptors are CD36 and CD163.

[0017] In some implementations, the integrin receptor is CD11b.

[0018] In some implementations, the C-type lectin receptor is a mannose receptor.

[0019] In some embodiments, the ligand is selected from one or more of the group consisting of dextran sulfate, oxidized or acetylated low-density lipoprotein, fucoidan sulfate, CpG ODN, mannose, galactose, hyaluronic acid, and anti-CD163 antibody, anti-CD36 antibody, anti-CD11b antibody, and anti-CD206 antibody.

[0020] In some implementations, the drug delivery carrier is a nanoparticle.

[0021] In some implementations, the drug delivery carrier is a liposome, a lipid nanoparticle, or a polymer nanoparticle.

[0022] In some implementations, the drug delivery carrier is a lipid nanoparticle.

[0023] In some implementations, the drug delivery carrier is mannose-modified lipid nanoparticles.

[0024] The beneficial effects of this invention are that by providing a composition comprising S100A9 antisense oligonucleotide and a drug delivery carrier specifically targeting macrophages, its application during the progression of liver fibrosis significantly improves liver function and reverses liver fibrosis, demonstrating a therapeutic effect superior to the S100A9 small molecule inhibitor pacquimod. This invention is the first to reveal the relationship between macrophage S100A9 protein and the development of liver fibrosis, and verifies in animal studies that precisely targeting liver macrophages to silence S100A9 protein expression can effectively reverse liver fibrosis, providing a reliable theoretical basis and potential technical means for the clinical treatment of liver fibrosis. Attached Figure Description

[0025] The accompanying drawings, which are provided to further illustrate embodiments of the invention and constitute a part of this invention, are not intended to limit the scope of the invention.

[0026] Figure 1The figure shows the bioinformatics analysis results of quantitative proteomics in liver tissue of fibrotic mice. In the figure, A represents protein analysis results; B represents GO-BP analysis results; and C represents protein-protein interaction network analysis results.

[0027] Figure 2 The image shows the results of immunohistochemistry and immunofluorescence colocalization of liver tissues from mice with CCl4-induced fibrosis for 4 and 8 weeks.

[0028] Figure 3 This image shows the co-localization results of immunohistochemistry and immunofluorescence in liver tissues from patients at different stages of clinical liver fibrosis and liver tissues from healthy controls.

[0029] Figure 4 The diagram shows the therapeutic effects of pacquimod on liver fibrosis in mice. A is a flowchart; B shows the results of H&E staining, Sirius red staining, and α-SMA immunohistochemical staining; C shows the serum ALT detection results; and D shows the serum AST detection results.

[0030] Figure 5 Figure 1 shows the results of single-cell transcriptome sequencing analysis of S100A9 expression distribution and changes in immune cells from liver tissue of fibrotic mice. In the figure, A represents the results of all cell analyses; B represents the results of myeloid cell analyses; and C represents the changes in S100A9 expression levels from different sources with CCl4 induction time.

[0031] Figure 6 The diagram shows the results of the myeloid S100A9 knockout experiment. A is a flowchart; B shows the neutrophil clearance results; and C shows the H&E staining, Sirius red staining, and α-SMA immunohistochemical staining results.

[0032] Figure 7 Figure 1 shows the S100a9-ASO sequence screening process and the results of validating the effect of interfering with S100A9 protein. In the figure, A is the S100a9-ASO sequence screening flowchart; B is the immunoblotting result of S100A9 expression in 293T cells; C is the result of real-time quantitative PCR detection of S100A9 transcriptional level; and D is the result of immunoblotting detection of S100A9 protein level.

[0033] Figure 8 pHAGE- S100a9 Figures showing plasmid construction and sequencing verification results. In the figures, A is a schematic diagram of plasmid construction; B is the colony PCR verification result; and C and D are the sequencing verification results.

[0034] Figure 9 For LNP- S100a9Figure 1 shows the results of a mouse liver fibrosis treatment model using the -ASO delivery system. A is a flowchart; B shows the immunoblotting results; C shows the H&E staining, Sirius red staining, and α-SMA immunohistochemical staining results; D shows the serum ALT detection results; and E shows the serum AST detection results. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood in the technical field to which this invention pertains. The following definitions are supplementary to those definitions in the art and relate to this invention, but are not extrapolated to any relevant or unrelated situation, such as any conventionally used patent or application. While any methods and materials similar to or equivalent to those described herein may be used in practical testing of the invention, the materials and methods described herein are preferred. Therefore, the terminology used herein is intended to describe specific embodiments only and is not intended to limit the invention.

[0037] The terms "comprising," "including," and "having" in this invention are open-ended descriptions, encompassing the specified steps described, as well as other steps that do not materially affect them, and are optional and not excluded. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist of said sequence, or may have additional amino acids or nucleotides at one or both ends of said protein or nucleic acid, but still have the same or similar activity as the original sequence.

[0038] As mentioned above, in response to the limited means of clinical treatment for liver fibrosis and the current lack of approved drugs for the treatment of liver fibrosis, the inventors completed this invention after a series of studies.

[0039] The present invention provides the use of a composition in the preparation of a medicament for the treatment of liver fibrosis, the composition comprising an S100A9 antisense oligonucleotide and a drug delivery carrier specifically targeting macrophages, the drug delivery carrier being capable of loading the S100A9 antisense oligonucleotide.

[0040] As used in this invention, "antisense oligonucleotides" (ASOs) refer to oligonucleotides that can induce gene silencing. Generally, ASOs are 15-22 nucleotides in length and are designed to be anticomplementary to the target RNA, hence the name antisense oligonucleotides. Antisense oligonucleotides can bind to the target RNA, forming an RNA / DNA heterodimer, which is then recognized and degraded by endogenous cellular RNase H, thereby inducing gene silencing. Furthermore, after binding to the target mRNA, they prevent ribosomes from binding to the mRNA, thus preventing the mRNA from being translated into the corresponding protein.

[0041] In some implementations, the S100A9 antisense oligonucleotide is selected from SEQ ID NO:2 and SEQ ID NO:3.

[0042] SEQ ID NO:2:

[0043] (2-O-MOE)C*(2-O-MOE)U*(2-O-MOE)G*(2-O-MOE)A*(2-O-MOE)A*T*T*C*C*T*T*C*T*T *G*(2-O-MOE)C*(2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)A*(2-O-MOE)G; where MOE is methoxyethyl modification.

[0044] SEQ ID NO:3:

[0045] (2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)U*T*T*C*T*T*C*A*T*A *A*(2-O-MOE)A*(2-O-MOE)G*(2-O-MOE)G*(2-O-MOE)U*(2-O-MOE)U; where MOE is methoxyethyl modification.

[0046] In some implementations, the S100A9 antisense oligonucleotide is SEQ ID NO:2.

[0047] In some implementations, the drug delivery vehicle targets macrophages through the interaction of a ligand and a receptor.

[0048] It is understandable that, based on the receptors specific to the surface of macrophages, the delivery vector is loaded with ligands that specifically bind to the receptors, thereby targeting macrophages.

[0049] In some implementations, the drug delivery vehicle targets one or more receptors on macrophages selected from the group consisting of scavenger receptors, Toll-like receptors, integrin receptors, and C-type lectin receptors.

[0050] In some implementations, the scavenger receptors are CD36 and CD163.

[0051] In some implementations, the integrin receptor is CD11b.

[0052] In some implementations, the C-type lectin receptor is a mannose receptor.

[0053] In some embodiments, the ligand is selected from one or more of the group consisting of dextran sulfate, oxidized or acetylated low-density lipoprotein, fucoidan sulfate, CpG ODN, mannose, galactose, hyaluronic acid, anti-CD163 antibody, anti-CD36 antibody, anti-CD11b antibody, and anti-CD206 antibody.

[0054] In some implementations, the drug delivery carrier is a nanoparticle.

[0055] In some implementations, the drug delivery carrier is a liposome, a lipid nanoparticle, or a polymer nanoparticle.

[0056] In some implementations, the drug delivery carrier is a lipid nanoparticle.

[0057] In some implementations, the drug delivery carrier is mannose-modified lipid nanoparticles.

[0058] The following describes preferred embodiments of the present invention, but the scope of protection of the present invention is not limited to these preferred embodiments. It should be noted that any modifications and improvements made by those skilled in the art based on this inventive concept are within the scope of protection of the present invention. All reagents used, unless otherwise specified, are commercially available conventional products.

[0059] Example 1: Identification of S100A9, a key differentially expressed protein in liver fibrosis

[0060] Six- to eight-week-old male C57BL / 6J mice (Beijing Spefol Biotechnology Co., Ltd.) were selected and divided into experimental and control groups (n=6 per group). A liver fibrosis induction reagent was prepared using a CCl4 to olive oil (sterilized at high temperature) ratio of 1:7 and used immediately. Each mouse in the experimental group was injected with 200 μL of the liver fibrosis induction reagent; each mouse in the control group was injected with 200 μL of olive oil. Injections were administered three times a week for 12 weeks. After 12 weeks, liver tissue was harvested, flash-frozen in liquid nitrogen, and stored at -80°C. Quantitative proteomics studies using tandem mass spectrometry tagging (TMT) were performed on the obtained liver tissue, yielding deep-coverage proteomics data of mouse liver tissue.

[0061] like Figure 1 As shown in A, P <0.05, and a fold change (comparison of CCl4-induced experimental group and olive oil control group) >2 were used as screening criteria. A total of 109 upregulated proteins and 69 downregulated proteins were identified.

[0062] like Figure 1 As shown in Figure B, gene ontology-biological process (GO-BP) enrichment analysis of 109 upregulated proteins revealed significant enrichment in processes associated with inflammatory responses.

[0063] Existing research indicates that the development and progression of liver fibrosis are inextricably linked to liver tissue inflammation. Therefore, further protein-protein interaction network analysis was performed on all inflammation-related proteins involved in the pathway, and the results are as follows: Figure 1 As shown in Figure C, the S100 calcium-binding protein A9 (S100A9) is located at the central node of the interaction network.

[0064] In summary, the results indicate that the abnormal upregulation of S100A9 protein in liver tissue is a significant event in the development and progression of liver fibrosis and has the potential to serve as a target.

[0065] Example 2: S100A9 detection in liver fibrosis tissue

[0066] Liver tissues from mice with CCl4-induced fibrosis for 4 and 8 weeks, and liver tissue pathological sections from patients at different clinical stages of liver fibrosis (stage S1, stage S2, and cirrhosis) were collected (provided by the Department of Pathology, Fifth Hospital of Shijiazhuang City) to verify the expression and distribution of S100A9 protein. The specific experimental procedure is as follows:

[0067] Immunohistochemical staining:

[0068] (1) Dewaxing and hydration: Place the paraffin sections in an oven at 55°C and bake for 1 hour. Perform dewaxing treatment according to the following steps: immerse the sections in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol for 2 minutes, 95% ethanol for 2 minutes, 80% ethanol for 2 minutes, 70% ethanol for 2 minutes, and finally wash twice with distilled water for 5 minutes each time.

[0069] (2) Blocking endogenous peroxidase: Place the slices in a humidified chamber, add 3% hydrogen peroxide solution, incubate at room temperature in the dark for 15 min, wash with PBS 3 times, 5 min each time.

[0070] (3) Antigen retrieval: Tissue sections were placed in a retrieval box and filled with 0.01 mol / L sodium citrate buffer to cover the tissue. Antigen retrieval was performed in a microwave oven on medium-high heat for 10 minutes until the liquid boiled. After natural cooling, the tissue sections were washed three times with PBS for 5 minutes each time.

[0071] (4) Blocking with normal goat serum: After the section is dried, wipe the liquid around the tissue, add normal goat serum to the tissue, place it flat in a humidified box, and incubate at 37°C for 30 min.

[0072] (5) Primary antibody incubation: Spin the slice dry, wipe off the surrounding blocking solution, add primary antibody (anti-S100A9 rabbit monoclonal antibody diluted with normal goat serum at a ratio of 1:1000), place it flat in a humidified box and incubate at room temperature for 30 min.

[0073] (6) Secondary antibody incubation: Wash with PBS 3 times, 5 min each time. Shake the sections dry, wipe off the liquid around the tissue, add secondary antibody (horseradish peroxidase conjugated goat anti-rabbit IgG polymer), evenly cover the tissue, place flat in a humidified box and incubate at 37°C for 30 min.

[0074] (7) DAB staining: Wash with PBS 3 times, 5 min each time. After shaking off the liquid around the tissue, add DAB staining solution (prepared according to the kit instructions, freshly prepared). Control the staining time under a microscope. A positive reaction is brownish-yellow. Shake off the DAB staining solution and immerse the slide in PBS to stop the staining.

[0075] (8) Counterstaining cell nuclei: Counterstain with hematoxylin for 20 seconds, rinse with tap water for 15 minutes to return to blue.

[0076] (9) Dehydration and mounting: The slides were placed in 80% ethanol for 2 min, 95% ethanol for 2 min, anhydrous ethanol for 10 min, xylene II for 5 min, and xylene I for 5 min in sequence to dehydrate and clear them. After drying, they were mounted with neutral resin.

[0077] (10) Microscopic examination (Nikon Ni-e) and image acquisition and analysis under 10x objective lens. Hematoxylin stained the cell nucleus blue and the DAB-positive area was brownish-yellow.

[0078] Immunofluorescence co-localization:

[0079] Similar to immunohistochemical staining, (1)-(4) the tissue sections were dewaxed and hydrated, endogenous peroxidase was blocked, antigen retrieval was performed, and serum was blocked. Subsequent steps are as follows:

[0080] (5) Primary antibody incubation: Pour off the serum, wipe the surrounding blocking solution dry, add the primary antibody (anti-S100A9 rabbit monoclonal antibody diluted with normal goat serum at a ratio of 1:1000), and incubate overnight at 4°C in a humidified box.

[0081] (6) Secondary antibody incubation: Wash 3 times with PBS, 5 min each time. Add horseradish peroxidase-conjugated goat anti-rabbit IgG polymer and incubate at 37°C for 30 min.

[0082] (7) Tyrosamide signal amplification (TSA) technique: Dilute fluorescein isothiocyanate (FITC) dye with TSA color development solution at a ratio of 1:100, drop it onto the tissue, let it stand in the dark for 1 min, and then soak it in distilled water for 5 min to terminate the color development.

[0083] (8) Elution: Add elution solution to the tissue and incubate at 37°C for 30 min to remove the bound primary and secondary antibodies.

[0084] (9) Soak in PBS for 5 min, and repeat steps (5)-(7) to complete the staining of mouse macrophage marker F4 / 80 or human macrophage marker CD68.

[0085] (10) 4′,6-Diamidinyl-2-phenylindole (DAPI) staining: DAPI is a fluorescent dye that can bind strongly to DNA. DAPI dye and PBS were diluted at a ratio of 1:500 and added to the tissue. The tissue was incubated at room temperature for 10 min. After rinsing with PBS, the tissue was mounted with glycerol mounting medium and examined under a super-resolution confocal microscope (Nikon A1R). Image acquisition and analysis were performed under a 20x objective lens.

[0086] result:

[0087] The results of immunohistochemical and immunofluorescence colocalization of liver tissues from CCl4-induced fibrotic mice at 4 and 8 weeks of fibrosis are as follows: Figure 2As shown, with increasing CCl4 induction time, i.e., with the development of liver fibrosis, the expression of S100A9 in liver tissue gradually increases, mainly in the central vein, portal area, and other major fibrotic areas. Immunofluorescence colocalization analysis results show that S100A9 (green fluorescence) and macrophage marker F4 / 80 (red fluorescence) colocalize, and their number gradually increases with the aggravation of liver fibrosis (e.g., ...). Figure 2 (As shown by the white arrow in the immunofluorescence co-localization map).

[0088] The results of immunohistochemical and immunofluorescence colocalization of liver tissues from patients at different stages of liver fibrosis (stage S1, stage S2, and cirrhosis) and healthy controls are as follows: Figure 3 As shown, with the progression of liver fibrosis, the expression of S100A9 (green fluorescence) increases significantly and continuously. It is also highly expressed in fibrotic areas such as the central vein and portal areas, and exhibits significant co-localization with the human macrophage marker CD68 (e.g., Figure 3 (As shown by the white arrows in the immunofluorescence co-localization map). Notably, S100A9 was significantly upregulated in the early stages of liver fibrosis, namely clinical stages S1 and S2. These results indicate that the abnormal upregulation of macrophage S100A9 in fibrotic liver tissue is species-conserved and may be involved in the early development of liver fibrosis.

[0089] Example 3: The therapeutic effect of S100A9 inhibitor pacquimod on liver fibrosis in mice

[0090] like Figure 4As shown in Figure A, a mouse liver fibrosis model induced by CCl4 for 4 weeks was constructed as described in Example 1. Mice were divided into a control group (Oil, N=5), a model group (Veh, N=5), a low-dose Paquinimod (PAQ) group (PAQ 2 mg / kg, N=4), and a high-dose Paquinimod group (PAQ 5 mg / kg, N=3). Each mouse in the control group was injected with 200 μL of olive oil three times a week for 4 weeks. Mice in the model group were injected with CCl4 for one week, followed by an equal volume of solvent during the next three weeks of CCl4 injection. Mice in the low-dose group were injected with CCl4 for one week, followed by 2 mg / kg Paquinimod during the next three weeks of CCl4 injection. Mice in the high-dose group were injected with CCl4 for one week, followed by 5 mg / kg Paquinimod during the next three weeks of CCl4 injection. After modeling, mice were sacrificed, serum was obtained, and liver tissue was used to assess liver fibrosis. The levels of serum liver injury markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using a fully automated biochemical analyzer (Hitachi 7020). Hematoxylin and eosin (H&E) staining and Sirius red staining (red staining area indicates collagen fibers) were performed at Wuhan Saiweier Biotechnology Co., Ltd. The immunohistochemical staining of liver fibrosis marker α-smooth muscle actin (α-SMA) was performed according to the steps in Example 2.

[0091] like Figure 4 As shown in Figures B, C, and D, after 4 weeks of CCl4 induction, compared with the Oil control group, the model group mice exhibited severe liver tissue damage, disordered hepatocyte arrangement, increased inflammatory cell infiltration, collagen deposition, and significantly increased α-SMA staining positive area, serum ALT, and AST levels. Different doses of pacquimod showed no amelioration effect on liver damage or liver fibrosis in the mice.

[0092] Example 4: Identification of the source of key S100A9 cells in fibrotic liver tissue

[0093] The origin of S100A9 cells was analyzed using the public single-cell transcriptome sequencing dataset CRA003280. This dataset contains CD45-positive cells from mouse liver fibrosis tissue induced by CCl4 at different time points, representing a population of hepatic immune cells.

[0094] like Figure 5 As shown in Figure A, all cells are divided into four major groups: B lymphocytes, T lymphocytes, NK cells, and myeloid cells. It can be seen that S100A9 is expressed at the highest level in myeloid cells.

[0095] Next, as Figure 5As shown in Figure B, myeloid cells are subdivided into neutrophils, monocytes / macrophages, and dendritic cells (where the size of the circle indicates the proportion of cells expressing S100A9 in that cell group, and the color intensity indicates the average expression level of S100A9 in a certain cell group). It can be seen that the expression proportion and average expression level of S100A9 are most significant in neutrophils, which is consistent with previous reports.

[0096] Further analysis was conducted on the changes in S100A9 expression levels from different sources with CCl4 induction time, such as... Figure 5 As shown in Figure C, monocyte / macrophage-specific S100A9 expression was continuously upregulated with prolonged CCl4 induction time, while neutrophil-specific S100A9 expression remained unchanged or even decreased. This suggests that macrophage-derived S100A9 may play a unique role in the development and progression of liver fibrosis.

[0097] Example 5: Myeloid knockout of S100A9 alleviates liver fibrosis in mice, independent of neutrophils.

[0098] Using the Cre-loxP recombinase system, myeloid-specific knockout mice of S100A9 (genotype: S100A9 fl / fl , Lyz2 -cre + ), S100A9 fl / fl As littermate control mice. Figure 6 As shown in Figure A, a mouse liver fibrosis model induced by CCl4 for 3 weeks was constructed using the same method as in Example 1. S100A9 fl / fl , Lyz2 -cre + (N=5) and S100A9 fl / fl (N=5) Both groups of mice were administered Ly6G neutralizing antibody concurrently with CCl4 injection to eliminate neutrophils. Sampling and liver fibrosis assessment were performed as described in Example 3. Peripheral blood neutrophils (CD11b) were detected by flow cytometry. + Ly6G + This represents the clearance of neutrophils.

[0099] The results are as follows Figure 6 As shown in Figures B and C, when neutrophils were effectively removed, collagen deposition and the positive area of ​​α-SMA staining in myeloid S100A9 knockout mice were significantly reduced, indicating that myeloid-specific S100A9 knockout inhibits CCl4-induced liver fibrosis in mice, and this inhibition is independent of the role of neutrophil S100A9.

[0100] Example 6: Targeting macrophage S100A9 effectively treats liver fibrosis in mice

[0101] S100A9 antisense oligonucleotide sequence screening:

[0102] against S100A9 Gene sequences were designed, and five ASOs (numbered ASO-1-5) were synthesized at Beijing Qingke Biotechnology Co., Ltd. The sequences are as follows:

[0103] SEQ ID NO:1:

[0104] (2-O-MOE)U*(2-O-MOE)G*(2-O-MOE)A*(2-O-MOE)U*(2-O-MOE)G*G*A*A*G*G* T*G*T*C*G*(2-O-MOE)A*(2-O-MOE)U*(2-O-MOE)G*(2-O-MOE)A*(2-O-MOE)U;

[0105] SEQ ID NO:2:

[0106] (2-O-MOE)C*(2-O-MOE)U*(2-O-MOE)G*(2-O-MOE)A*(2-O-MOE)A*T*T*C*C*T* T*C*T*T*G*(2-O-MOE)C*(2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)A*(2-O-MOE)G;

[0107] SEQ ID NO:3:

[0108] (2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)U*T*T*C*T*T* C*A*T*A*A*(2-O-MOE)A*(2-O-MOE)G*(2-O-MOE)G*(2-O-MOE)U*(2-O-MOE)U;

[0109] SEQ ID NO:4:

[0110] (2-O-MOE)A*(2-O-MOE)A*(2-O-MOE)G*(2-O-MOE)C*(2-O-MOE)U*C*A*G*C*T* G*A*T*T*G*(2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)C*(2-O-MOE)U*(2-O-MOE)G;

[0111] SEQ ID NO:5:

[0112] (2-O-MOE)U*(2-O-MOE)G*(2-O-MOE)U*(2-O-MOE)U*(2-O-MOE)C*T*C*A*T*G* C*A*G*C*T*(2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)U*(2-O-MOE)C*(2-O-MOE)A;

[0113] MOE is modified with methoxyethyl.

[0114] The S100A9 interference effect was verified on five sequences. The procedure is as follows: Figure 7 As shown in Figure A: Constructing the pHAGE-S100A9 expression plasmid → Constructing 293T cells transiently overexpressing S100A9 (293T S100A9-OE → Transfection with S100A9-ASO → Detection of S100A9 transcriptional and protein levels.

[0115] (1) pHAGE- S100A9 Plasmid construction:

[0116] like Figure 8 As shown in Figure A, homologous recombination was used to construct... S100A9 Expression plasmids are first designed using primers containing homologous arms, so that... S100A9 The 5' and 3' ends of the target fragment PCR product contain sequences that perfectly match the front and rear ends of the pre-inserted linearized pHAGE vector, respectively. Using mouse spleen cDNA as a template, [the following was performed / conducted]... S100A9 Primers: Upstream primer 5'-CACACTCAGATCATCTTCTCAA-3' (SEQ ID NO: 6), downstream primer 5'-GGACGTCGTATGGGTATTACTTCCCACAGCCTTTGC-3' (SEQ ID NO: 7), the target gene was amplified by PCR. The amplified products were separated by agarose gel electrophoresis, gel excision and purification, and the sequence was confirmed to be correct by sequencing. Simultaneously, using pHAGE primers: upstream primer 5'-TACCCATACGACGTCCCAGACTACGCTTAG-3' (SEQ ID NO: 8), downstream primer 5'-GGTGGCAAAGCTTAAGTTTAAACGCTAGCCA-3' (SEQ ID NO: 9), the pHAGE circular vector was amplified into a linearized vector fragment by PCR, and purified using the same method.

[0117] Will S100A9The fragment (800 ng) and pHAGE vector (400 ng) were mixed in a specific ratio and incubated at 37°C for 2 h using 2×CE Mix to complete recombination ligation. The ligation product was transformed into *E. coli* DH5α competent cells, incubated on ice for 3 min, then heat-shocked at 42°C for 90 s, and immediately cooled on ice for 2 min. 200 μL of antibiotic-free LB broth was added to the tube, and the cells were incubated at 37°C with shaking at 180 rpm for 1 h. 50 μL of the bacterial culture was spread onto LB agar plates containing ampicillin and incubated at 37°C for 16 h. Single colonies were picked for PCR verification (verification results are shown in the figure). Figure 8 As shown in Figure B), after the positive clones were expanded and cultured, part of the bacterial culture was stored at -80℃ with 80% glycerol, and the remaining bacterial culture was used to extract recombinant plasmids using a plasmid miniprep kit (DP103-03, TIANGEN). After sequencing confirmed that the recombinant plasmid sequence was correct (sequencing results are shown in Figure B), the recombinant plasmid was extracted. Figure 8 (As shown in C and D) are used for subsequent experiments.

[0118] (2)293T S100A9-OE Build

[0119] 293T cells were seeded at a density of 60% into six-well plates and cultured in DMEM complete medium for 12 h. The original medium was then replaced with Opti-MEM serum-depleted medium (#31985070, Thermo Fisher Scientific). 2.5 μg of pH AGE- S100A9 The plasmid was gently added to 150 μL of Opti-MEM, and simultaneously, 5 μL of X-tremeGENE HP DNATransfection Reagent (#06366236001, Roche) was gently added to the same 150 μL of Opti-MEM. The two were gently mixed and incubated at room temperature for 15 min to form a transfection complex. The transfection mixture was then evenly added to six-well plates and cultured for 6 h, after which the medium was replaced with DMEM complete medium. Cells were collected at 48 h and 72 h after transfection for Western blotting to detect S100A9 protein levels. Figure 7 As shown in B, pHAGE- S100A9 The S100A9 protein was successfully overexpressed in 293T cells 48 or 72 h after plasmid transfection.

[0120] (3) S100A9-ASO transfection

[0121] Transfection with pHAGE as described in (2) S100A9Plasmids were introduced into 293T cells, and after 24 hours, the medium was replaced with Opti-MEM medium containing reduced serum. Control or different sequences of S100A9-ASO were added to Opti-MEM, along with 5 μL of Lipo2000 transfection reagent (#11668019, Thermo Fisher Scientific). The mixture was gently mixed and incubated at room temperature for 15 minutes to form a transfection complex. The transfection complex was then slowly and evenly added to the corresponding wells. After 6 hours of culture, the medium was replaced with DMEM complete medium, and cells were cultured for another 18 hours. Cells were then harvested, and S100A9 transcriptional levels were detected by real-time quantitative PCR or S100A9 protein levels were detected by Western blotting.

[0122] Transcribed RNA levels such as Figure 7 As shown in Figure C, the protein expression level is as follows: Figure 7 As shown in Figure D, at the RNA level, ASO-2 has the best interference effect on S100A9, showing a significant difference compared to ASO-1 (one-way ANOVA). P The value was 0.0041, which showed no statistically significant difference from ASO-3 (one-way ANOVA). P (Value = 0.1687). At the protein level, ASO-2 showed the best silencing effect on the S100A9 protein. Based on the above results, the ASO-2 sequence was selected for subsequent experiments.

[0123] 2. Construction of mannose-modified lipid nanoparticles (LNP-) loaded with S100A9 antisense oligonucleotides. S100A9 A mouse model of liver fibrosis treatment using an ASO delivery system

[0124] Based on the above results, ASO-2 was selected as the optimal sequence and encapsulated within mannose-modified lipid nanoparticles, which were prepared by Nanjing Genscript Biotech Co., Ltd. The specific experimental procedure is as follows:

[0125] A mouse liver fibrosis model induced by CCl4 for 8 weeks was constructed as described in Example 1, and the mice were divided into four groups: olive oil group (Oil, N=3), olive oil + LNP-ASO administration group (Oil + LNP, N=3), CCl4 induction group (CCl4, N=6), and CCl4 induction + LNP-ASO administration group (CCl4 + LNP, N=5). Figure 9 As shown in Figure A, LNP-ASO administration began 4 weeks after induction with olive oil or CCl4, administered via tail vein injection twice weekly at a dose of 5 mg / kg mouse body weight. After another 4 weeks of induction, the mice were sacrificed, and tissue samples were collected and liver fibrosis was assessed as described in Example 3.

[0126] like Figure 9As shown in Figure B, after LNP-ASO treatment (hereinafter referred to as LNP), the immunoblotting results showed that the expression of S100A9 in the liver tissue of mice treated with LNP was significantly reduced compared with the untreated group, indicating that LNP treatment successfully targeted hepatic macrophages and silenced S100A9 expression in macrophages. Furthermore, as... Figure 9 As shown in C, D, and E, the liver fibrosis status of mice was assessed by histopathological staining. It can be seen that under CCl4 induction, mice without LNP administration showed severe damage to liver tissue structure, disordered hepatocyte arrangement, increased inflammatory cell infiltration, collagen deposition, and significantly increased α-SMA staining positive area, serum ALT and AST levels compared with the Oil control group.

[0127] LNP administration (CCl4+LNP) vs After CCl4 treatment, the liver tissue structure partially recovered, and collagen deposition [(0.67±0.13)%] vs (2.75±0.56)%, P Value <0.001], α-SMA positive staining area [(0.55±0.10)%] vs (3.43±0.70)%, P [Value < 0.001], serum ALT (1094.40 ± 250.53) vs 7040.00±3310.32 U / L P Value <0.01) and AST (651.20±105.01) vs 3889.33±1929.09 U / L P The levels of LNP (value <0.01) were significantly reduced. Notably, in the olive oil-induced control group mice, LNP administration did not cause pathological damage to the liver tissue, suggesting that LNP has a certain degree of safety.

[0128] These results combined demonstrate that LNP-targeted therapy during the advanced stage of liver fibrosis successfully reverses existing liver fibrosis and effectively alleviates liver damage.

[0129] This document uses specific embodiments to illustrate the principles and implementation schemes of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and central idea of ​​the present invention, and are not intended to limit the process method. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall under the protection of the claims of the present invention.

Claims

1. The use of the composition in the preparation of a medicament for treating liver fibrosis, characterized in that, The composition comprises S100A9 antisense oligonucleotide and a drug delivery vehicle that specifically targets macrophages. The drug delivery carrier can load the S100A9 antisense oligonucleotide, which is selected from the sequences shown in SEQ ID NO:2 and SEQ ID NO:

3.

2. The use as described in claim 1, characterized in that, The S100A9 antisense oligonucleotide is the sequence shown in SEQ ID NO:

2.

3. The use as described in claim 1, characterized in that, The drug delivery vehicle targets macrophages through the interaction of ligands and receptors.

4. The use as described in claim 3, characterized in that, The drug delivery vehicle targets one or more receptors on macrophages selected from the group consisting of scavenger receptors, Toll-like receptors, integrin receptors, and C-type lectin receptors.

5. The use as described in claim 3, characterized in that, The ligand is selected from one or more of the group consisting of dextran sulfate, oxidized or acetylated low-density lipoprotein, fucoidan sulfate, CpG ODN, mannose, galactose, hyaluronic acid, anti-CD163 antibody, anti-CD36 antibody, anti-CD11b antibody, and anti-CD206 antibody.

6. The use as described in claim 3, characterized in that, The drug delivery carrier is a nanoparticle.

7. The use as described in claim 3, characterized in that, The drug delivery carrier is of the type of liposome, lipid nanoparticle, or polymer nanoparticle.

8. The use as described in claim 3, characterized in that, The drug delivery carrier is a lipid nanoparticle.

9. The use as described in claim 3, characterized in that, The drug delivery carrier is mannose-modified lipid nanoparticles.

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