Use of prmt6 in the prevention and treatment of macrophage-mediated inflammatory response and pharmaceutical composition thereof

Abstract

The present application discloses the use of protein arginine methyltransferase 6 (PRMT6) in preventing and treating macrophage-mediated inflammatory response and its pharmaceutical composition, specifically discloses the use of PRMT6, its coding sequence or its promoter in preparing a drug for preventing and / or treating macrophage-mediated inflammatory response.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to the anti-macrophage-mediated inflammatory effects, mechanisms, implementation methods, and uses of protein arginine methyltransferase 6 (PRMT6) in inflammatory diseases (such as inflammatory bowel disease). Background Technology

[0002] Macrophages play a crucial role in maintaining intestinal homeostasis through immune regulation, barrier repair, and inflammation resolution (Hegarty et al., 2023). The pathogenesis of inflammatory bowel disease (IBD) is closely related to defects in macrophage phenotypic switching, dysregulation of environmental signaling, and genetic susceptibility (Na et al., 2019). In inflamed tissues, resident macrophages and recruited monocytes undergo phenotypic reprogramming through interactions with cytokines and pathogen- / damage-associated molecular patterns (PAMPs / DAMPs), ultimately differentiating into a large number of pro-inflammatory effector groups that aggregate in the pathological microenvironment (Di Benedetto et al., 2019; Pan et al., 2022). Notably, IL-1β secreted by activated macrophages is a key mechanism for the activation of the innate immune response and tissue destruction in IBD. Clinical observations and preclinical studies have confirmed that elevated IL-1β levels in patients with active IBD are associated with the severity of mucosal ulcers (Liso et al., 2022; Neurath, 2024). Inhibition of IL-1 function can improve the activity of experimental colitis and reduce the occurrence of inflammation-related carcinogenesis (Neurath, 2014). Treatment with anakinra to block IL-1 receptor function can suppress the activity of experimental colitis in mice (Liso et al., 2022). Therefore, a more comprehensive understanding of the molecular mechanisms of IL-1β overproduction and elucidating strategies for restricting IL-1β transcription in homeostatic macrophages to prevent pathological overexpression is crucial.

[0003] Macrophage inflammatory responses and cytokine production are precisely regulated by epigenetic mechanisms (Q. Zhang & Cao, 2019, 2021). New evidence elucidates the multi-level epigenetic regulation of IL-1β. Notably, hnRNPA2B1-mediated demethylation of IL-1β enhancer DNA 6mA has been shown to increase chromatin accessibility and activate IL-1β transcription upon immune stimulation (Shihao Zhang, 2025). Furthermore, recent studies have shown that H3K9 crotonylation directly regulates IL-1β expression through histone modification-dependent transcriptional regulation (Li et al., 2024). However, the dynamic regulatory mechanisms by which these epigenetic modifications strictly suppress IL-1β expression under homeostatic conditions and rapidly activate its transcription upon pathogen recognition remain unclear. This precise epigenetic intervention holds promise for circumventing the widespread immunosuppressive effects associated with traditional anti-cytokine therapies and potentially enabling targeted modulation of dysregulated inflammatory responses without compromising fundamental host defense mechanisms.

[0004] Trimethylation of histone H3 at position 4 (H3K4me3) is a marker of transcriptionally active chromatin, acting as a key epigenetic regulator of innate immune responses by controlling the accessibility and expression of inflammatory genes (Millán-Zambrano et al., 2022; Wang & Helin, 2024). This modification is primarily located at transcription start sites (TSSs), promoting transcription initiation and elongation by recruiting chromatin remodeling factors and basal transcriptional mechanisms (Jambhekar et al., 2019; Wang & Helin, 2024). In mammalian systems, H3K4me deposition is catalyzed by COMPASS family methyltransferases (SET1A / SET1B and MLL1-MLL4), which possess conserved SET catalytic domains but have different regulatory subunits, thus conferring target gene specificity (Albert & Kerl, 2023; Shilatifard, 2012). However, the molecular mechanisms that ensure precise spatiotemporal control of H3K4me3 deposition at inflammatory gene promoters, and the molecular mechanisms by which these enzymes are inhibited under equilibrium conditions to prevent abnormal hypermethylation of cytokine gene sites (such as IL-1β), remain not fully understood. Elucidating these mechanisms has significant therapeutic value for selectively resetting the pathogenic epigenetic state of chronic inflammation.

[0005] Protein arginine methyltransferase 6 (PRMT6), a type I arginine methyltransferase, catalyzes the asymmetric dimethylation of arginine at position 2 of histone H3 (H3R2me2a). This epigenetic marker enhances transcriptional silencing at target gene sites by antagonizing H3K4me2 / 3 deposition (Guccione et al., 2007; Hyllus et al., 2007; Kirmizis et al., 2007).

[0006] Although the role of PRMT6 in cancer, viral infection and osteoclast differentiation has been studied (Chu et al., 2024; Kong et al., 2024; Yu et al., 2024; H. Zhang et al., 2019), the function of this enzyme in maintaining immune homeostasis and suppressing autoimmune inflammation remains unknown.

[0007] In summary, there is an urgent need in this field to develop epigenetic regulators and related pharmaceutical compositions that can effectively inhibit macrophage-mediated inflammatory responses and prevent and / or treat infectious or autoimmune diseases. Summary of the Invention

[0008] To address the aforementioned technical problems, in one aspect, this application provides the use of PRMT6, its coding sequence, or an promoter thereof in the preparation of medicaments for the prevention and / or treatment of macrophage-mediated inflammatory responses.

[0009] In another aspect, this application provides the use of PRMT6, its coding sequence or promoter thereof in the preparation of medicaments for the prevention and / or treatment of infectious diseases or autoimmune diseases and their related conditions and / or symptoms.

[0010] In another aspect, this application provides a pharmaceutical composition comprising: (a) PRMT6, its coding sequence or its promoter; (b) A pharmaceutically or immunologically acceptable carrier or excipient; and (c) Other active ingredients used for the prevention or treatment of infectious diseases or autoimmune diseases and their related symptoms and / or signs. Attached Figure Description

[0011] The present application will now be described in more detail with reference to the accompanying drawings, in which: Figure 1 PRTM6-deficient mice exhibit increased macrophage-mediated inflammatory responses in the pathogenesis of colitis. A: Survival rate analysis; B: Mouse weight monitoring C: Monitoring of indications for colitis in mice D: Mouse intestinal length E: HE staining analysis; F: Semi-quantitative HE score G: Immunofluorescence analysis HJ: ELISA analysis of IL-1β, IL-6 and TNF-α; KM: Quantitative PCR analysis results targeting IL-1β mRNA; In the picture, "Indicates P < 0.05," "Indicates P < 0.01," " indicates P < 0.001.

[0012] Figure 2 PRTM6-deficient mice are more sensitive to endotoxins. A: Survival rate analysis; B: HE staining analysis; CE: ELISA analysis results of IL-1β, TNF-α and IL-6 in LPS; In the picture, "Indicates P < 0.05," "Indicates P < 0.01," " indicates P < 0.001.

[0013] Figure 3 PRTM6-deficient macrophages showed increased IL-1β production upon stimulation with LPS, Pam3CSK4, and poly(I:C). A: ELISA analysis results of IL-1β after LPS stimulation; B: Quantitative PCR analysis results of IL-1β after LPS stimulation; C: Quantitative PCR analysis results of IL-1β after Pam3CSK4 stimulation; D: Quantitative PCR analysis results of IL-1β after poly(I:C) stimulation; E: Results of Western blot analysis; In the picture, "Indicates P < 0.05," "Indicates P < 0.01," " indicates P < 0.001.

[0014] Figure 4 Macrophages overexpressing PRTM6 showed reduced IL-1β production upon stimulation with LPS, Pam3CSK4, and poly(I:C). A: Quantitative PCR analysis results of IL-1β in LPS, Pam3CSK4 and poly(I:C); B: Results of Western blot analysis; In the picture, "Indicates P < 0.05," "Indicates P < 0.01," " indicates P < 0.001.

[0015] Figure 5 PRMT6 promoter can alleviate colitis symptoms in mice.

[0016] AB: Expression analysis results of PRMT6; C: Body weight results in mice treated with KA; D: Results of KA treatment for enteritis symptoms in mice; E: Results of intestinal length in mice treated with KA; FG: Histopathological results of mice treated with KA; In the picture, "Indicates P < 0.05," "Indicates P < 0.01," " indicates P < 0.001. Detailed Implementation

[0017] This application relates to the use of PRMT6, its coding sequence, or promoters thereof in the preparation of medicaments for the prevention and / or treatment of macrophage-mediated inflammatory responses. In one embodiment, the inflammatory response is caused by a pathogen, a chemical substance, a physical factor, or a combination thereof. In one embodiment, the pathogen, chemical substance, physical factor, or combination thereof includes endotoxins, sodium dextran sulfate (DSS), LPS, Pam3CSK4, or poly(I:C). In one embodiment, the pathogen includes one or more selected from the group consisting of *Escherichia coli*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, *Acinetobacter baumannii*, and *Enterococcus faecalis*. In one embodiment, the inflammatory response includes inflammatory bowel disease, such as colitis. In one embodiment, the inflammatory response includes increased production of the inflammatory factors IL-1β, TNFα, and IL-6. In one embodiment, the prevention and / or treatment of macrophage-mediated inflammatory responses includes suppressing inflammation, maintaining immune homeostasis, improving organ function, and / or increasing patient survival.

[0018] As used herein, the terms “PRMT6,” “PRMT6 protein,” and “PRMT6 polypeptide” are used interchangeably to refer to protein arginine methyltransferase 6. PRMT6 may have a sequence known in the art, such as the sequence of SEQ ID NO: 1 or 3 or a protein encoded by a homologous sequence thereof, and may also include variant or modified forms of PRMT6. In one embodiment, PRMT6 may be selected from: (i) the amino acid sequence of SEQ ID NO: 2 or 4; or (ii) an amino acid sequence having at least 80% identity with SEQ ID NO: 2 or 4. In one embodiment, (ii) includes an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 2 or 4. In one embodiment, PRMT6 may also be selected from: (iii) proteins or polypeptides from the amino acid sequences of (i) or (ii) that have been substituted, deleted, or added to one or more amino acids. In one embodiment, a protein or polypeptide whose amino acid sequence of (i) or (ii) has been substituted, deleted, or added with one or more amino acids includes a protein or polypeptide having substituted, deleted, or added amino acids of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids. In one embodiment, the amino acid substitution includes conserved substitution. In one embodiment, the amino acid sequence of (ii) and / or (iii) has activity that inhibits inflammatory responses. In one embodiment, PRMT6 may comprise a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher animals, insects, and mammalian cells) using recombinant technology. In a preferred embodiment, PRMT6 is encoded by the human PRMT6 gene or its homologs or family genes. In one embodiment, PRMT6 may be derived from mammals, such as humans or mice. In one embodiment, variations of PRMT6 include, but are not limited to, the deletion, insertion, and / or substitution of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties typically does not alter the function of the protein or peptide. Similarly, the addition of one or more amino acids at the C-terminus and / or N-terminus typically does not alter the function of the protein or peptide; for example, PRMT6 of this application may or may not include the initiating methionine residue while still possessing activity that inhibits macrophage-mediated inflammatory responses. In one embodiment, random mutagenesis can be induced by radiation or exposure to a mutagen, or the proteins or peptides described in (iii) above can be obtained by site-directed mutagenesis or other known molecular biology techniques.In one embodiment, a transgenic animal can be constructed using the coding sequence encoding the protein or polypeptide, and the resulting protein or polypeptide can be screened and identified by observing whether the transgenic animal's resistance to macrophage-mediated inflammatory responses is improved. In one embodiment, the protein or polypeptide of this application may be glycosylated or non-glycosylated, depending on the host used in the recombinant production protocol. In one embodiment, PRMT6 further includes an active fragment and an active derivative of the PRMT6 protein. In one embodiment, variants of PRMT6 include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by sequences that hybridize to the PRMT6 protein coding sequence under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the PRMT6 protein. In one embodiment, other polypeptides may also be used, such as fusion proteins containing the PRMT6 protein or fragments thereof. In one embodiment, PRMT6 may also include a soluble fragment of the PRMT6 protein. Typically, the fragment has at least about 10 consecutive amino acids of the PRMT6 protein sequence, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0019] As used herein, the terms “PRMT6 gene,” “PRMT6 coding sequence,” or “PRMT6 protein coding sequence” are used interchangeably and all refer to the nucleotide sequence encoding the PRMT6 described in this application. In one embodiment, the PRMT6 coding sequence may be selected from: (i) the nucleotide sequence of SEQ ID NO: 1 or 3; or (ii) a nucleotide sequence having at least 80% identity with SEQ ID NO: 1 or 3. In one embodiment, (ii) includes a nucleotide sequence having at least 85%, 90%, 95%, 98%, or 99% identity with SEQ ID NO: 1 or 3. In one embodiment, the PRMT6 coding sequence may also be selected from: (iii) a molecule that hybridizes to the nucleotide sequence of (i) or (ii) under stringent conditions, or a sequence that is highly homologous to said molecule (e.g., at least 80% identity). In one embodiment, the PRMT6 coding sequence comprises cDNA or mRNA. In one embodiment, the PRMT6 coding sequence has activity that inhibits macrophage-mediated inflammatory responses. In one embodiment, the PRMT6 coding sequence has activity that promotes phagocytosis and clearance of bacteria. In one embodiment, the PRMT6 gene may include the sequence shown in Gene ID: 72287 or MGI: 1919537, or a homologous sequence thereof, or a sequence modified therefrom. In one embodiment, the PRMT6 coding sequence may be derived from a mammal, such as a human or a mouse. As used herein, the term “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 × SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, more preferably 95%. In one embodiment, the full-length nucleotide sequence of the PRMT6 gene or a fragment thereof can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this application, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order. In a preferred embodiment, the PRMT6 coding sequence is obtained from a human.In one embodiment, the PRMT6 coding sequence includes other genes obtained from other animals that are highly homologous to the human PRMT6 gene (e.g., having 50% or more, preferably 55%, 60%, 65%, 70%, 75%, 80%, more preferably 85%, such as 85%, 90%, 95%, 98%, or even 99% or more of the sameness). Methods and tools for comparing sequence identity are also well known in the art, such as BLAST. In this application, "homology" and "identity" are used interchangeably.

[0020] As used herein, the terms "PRMT6 promoter" or "PRMT6 coding sequence promoter" are used interchangeably and refer to substances that can increase the level and / or activity of PRMT6 or its coding sequence. In one embodiment, a PRMT6 promoter includes substances that increase PRMT6 protein levels or promote PRMT6 function. In one embodiment, a PRMT6 promoter includes, but is not limited to: PRMT6 expression vectors, naked DNA encoding the PRMT6 coding sequence, liposome-encapsulated DNA encoding the PRMT6 coding sequence, LAFP precursor proteins or conjugates or complexes capable of being converted into PRMT6 in vivo, PRMT6 agonists, or combinations thereof. In one embodiment, a PRMT6 promoter has activity that inhibits macrophage-mediated inflammatory responses. In one embodiment, a PRMT6 promoter includes kamebakaurin.

[0021] This application also relates to the use of PRMT6, its coding sequence, or its promoter in the preparation of medicaments for the prevention and / or treatment of infectious diseases or autoimmune diseases and their related symptoms and / or signs. In one embodiment, PRMT6, its coding sequence, or its promoter may include PRMT6, its coding sequence, or its promoter as described in any of the foregoing embodiments. In one embodiment, PRMT6, its coding sequence, or its promoter has activity in inhibiting infectious diseases or autoimmune diseases and their related symptoms and / or signs. In one embodiment, the related symptoms and / or signs include one or more selected from the group consisting of diarrhea, hematochezia, excessive production of inflammatory factors, inflammatory shock or death, inflammatory damage to organs, and multiple organ failure. In one embodiment, the inflammatory factors include one or more selected from the group consisting of IL-1β, TNFα, and IL-6. In a preferred embodiment, the inflammatory factor includes IL-1β. In one embodiment, the organ includes a digestive organ. In one embodiment, the organ is selected from the intestine and spleen. In one embodiment, the therapeutic and / or preventive effects include promoting macrophage phagocytosis and clearance of bacteria.

[0022] This application also relates to a pharmaceutical composition comprising: (a) PRMT6, its coding sequence or an promoter thereof; (b) a pharmaceutically or immunologically acceptable carrier or excipient; and (c) other active ingredients for the prevention or treatment of macrophage-mediated inflammatory responses, infectious diseases or autoimmune diseases and their related conditions and / or symptoms. In one embodiment, PRMT6, its coding sequence or an promoter thereof may include PRMT6, its coding sequence or an promoter thereof as described in any of the preceding embodiments. In one embodiment, other active ingredients include biologics and / or small molecule drugs. In a preferred embodiment, other active ingredients include one or more selected from the group consisting of 5-aminosalicylic acid compounds, corticosteroids and immunosuppressants. In one embodiment, PRMT6, its coding sequence or an promoter thereof constitutes 0.001-99.9 wt%, for example 1-95 wt%, preferably 5-90 wt%, more preferably 10-80 wt% of the total weight of the pharmaceutical composition. In one embodiment, the balance is a pharmaceutically acceptable carrier and other additives. In one embodiment, (a) comprises an effective amount of PRMT6, its coding sequence or an promoter thereof. As used herein, the term "effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. In one embodiment, the pharmaceutical composition can be used to treat or prevent infectious diseases known in the art, such as the phagocytosis and clearance of bacteria in the body following bacterial infection; endotoxic shock or death; inflammatory damage to organs; and multiple organ failure. As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. As used herein, the term "pharmaceuticalally or immunologically acceptable carrier or excipient" refers to a carrier or excipient used for the administration of a therapeutic agent, including various diluents. This term refers to pharmaceutical carriers that are not themselves essential active ingredients and do not cause excessive toxicity after administration. Suitable carriers or excipients are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (MackPub. Co., NJ 1991). In one embodiment, an acceptable carrier or excipient may include liquids such as water, saline, glycerol, and ethanol. In another embodiment, an acceptable carrier or excipient may also include auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, and more preferably about 7.As used herein, the term "unit dosage form" refers to a dosage form prepared for a single dose of the pharmaceutical composition of this application for ease of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations. In another preferred embodiment, the pharmaceutical composition is a unit dosage form or multiple dosage forms, wherein the content of PRMT6, its coding sequence, or its promoter may be 0.01-2000 mg / dose, preferably 0.1-1500 mg / dose, more preferably 1-1000 mg / dose. In another preferred embodiment, 1-6 doses of the pharmaceutical composition are administered daily, preferably 1-3 doses; most preferably, the daily dose is 1 dose. It should be understood that the effective dose of PRMT6, its coding sequence, or its promoter may vary depending on the subject to be administered or treated. The specific situation depends on the individual circumstances of the subject (e.g., subject weight, age, physical condition, desired effect), which is within the scope of judgment of those skilled in the art. In one embodiment, the pharmaceutical composition may be solid (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid) or other suitable form. In one implementation, the administration route may be: (1) direct naked DNA or protein injection; (2) linking PRMT6 cDNA, mRNA and protein with transferrin / poly-L-lysine complex to enhance its biological effects; (3) forming complexes of cDNA, mRNA and protein with positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) encapsulating cDNA, mRNA and protein with liposomes to mediate their entry into the cell, which is conducive to the smooth entry of macromolecules and avoids hydrolysis by various extracellular enzymes; (5) binding cDNA, mRNA and protein with cholesterol increases their cytoplasmic retention time by 10 times; (6) transporting cDNA, mRNA and protein with immunoliposomes can specifically transport them to target tissues and target cells; (7) transfecting cDNA, mRNA and protein in vitro to regenerating cells (such as fibroblasts) can also better load PRMT6-related drugs into target cells; (8) electroporation, that is, introducing cDNA, mRNA and protein into target cells with the help of electric current. In one embodiment, the pharmaceutical composition is suitable for administration by means selected from the group consisting of: oral administration, injection (e.g., direct naked DNA or protein injection, liposome-coated DNA or protein injection), gold-coated gene gun bombardment, plasmid DNA carried by replication-defective bacteria, target DNA carried by replication-defective adenovirus, or protein encoded by a target gene, or electroporation. In one embodiment, the pharmaceutical composition may also contain other active ingredients for improving and treating bacterial infectious diseases.In one embodiment, the other active ingredients are selected from one or more of the following groups: clinically commonly used antibiotics (including β-lactams (penicillins and cephalosporins), aminoglycosides, tetracyclines, chloramphenicol, macrolides, antifungal antibiotics, and antituberculosis antibiotics). In one embodiment, PRMT6-related nucleotide and protein drugs can be used in combination with each other, and can also be combined with other drugs and treatments for the prevention and treatment of infectious diseases (e.g., bacterial infections).

[0023] This application may also relate to vectors containing the PRMT6 gene, host cells genetically engineered using such vectors, and transgenic animals that express high levels of PRMT6. In one embodiment, the PRMT6 coding sequence of this application can be used to express or produce recombinant PRMT6 protein using conventional recombinant DNA techniques (Science, 1984; 224:1431). Generally, the steps are as follows: (1) transforming or transducing suitable host cells with the polynucleotide (or variant) encoding the PRMT6 protein of this application, or with a recombinant expression vector containing such polynucleotide; (2) culturing the host cells in a suitable culture medium; and (3) isolating and purifying the protein or polypeptide from the culture medium or cells. In this application, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements. Methods well known to those skilled in the art can be used to construct expression vectors containing the PRMT6 coding sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In this application, pcDNA3.1 vectors, pIRES2-EGFP vectors, Adeno-X expression systems, etc., can be used. Furthermore, the expression vector may also contain one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli. Vectors containing the above-mentioned suitable DNA sequences and suitable promoters or control sequences can be used to transform suitable host cells to enable them to express proteins or peptides. In one embodiment, the host cell may include prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; and animal cells. In one embodiment, the host cell may include mouse macrophages. When the polynucleotides of this application are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. An enhancer is a cis-acting factor of DNA, typically approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.In this application, the terms "transgenic animal" or "transformed animal" are used interchangeably and refer to cells, organs, tissues, or individuals that have been transfected with the PRMT6 coding sequence of this application and stably and highly express PRMT6, obtained through conventional transgenic methods. The recombinant polypeptides in the above methods can be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0024] Compared with the prior art, the present invention has the following beneficial effects: Through extensive research and cell and animal model experiments, the inventors discovered that PRMT6 is a key epigenetic regulator of macrophage-mediated inflammation, effectively inhibiting inflammation, maintaining immune balance, improving organ function, and increasing patient survival rates in inflammatory bowel disease (IBD). Based on this, the inventors completed this application. Therefore, this application discloses novel functions of PRMT6, its coding sequence, and its promoters: inhibiting macrophage-mediated inflammatory responses in IBD, maintaining immune balance, protecting organ function, and improving the survival rate of affected individuals. This application also provides a novel drug that can effectively restore immune balance in individuals with IBD and improve the survival rate of infected individuals.

[0025] Specifically, applied research on genes that affect the phagocytic and clearance functions of macrophages is a hot topic in molecular and cell biology research. Applying nucleotides and proteins of genes that promote phagocytosis and clearance to the prevention and treatment of infectious diseases is an effective technique, and therefore has broad application prospects in both functional genomics research and related gene therapy.

[0026] The inventors discovered through research that PRMT6 deficiency significantly weakens the ability of mouse macrophages to phagocytose and clear bacteria. This was observed in LPS and Escherichia coli (…). E. coli In the inflammatory response induced by PRMT6, PRMT6 deficiency was observed to significantly reduce the production of pro-inflammatory cytokines and type I interferon by macrophages. PRMT6-deficient mice showed weaker clearance of E. coli and were more susceptible to E. coli infection. Furthermore, PRMT6 not only promotes endocytosis and bacterial killing but also maintains a moderate inflammatory response in the body. These findings suggest that PRMT6 may have potential applications in treating diseases such as bacterial infections. Therefore, this application provides methods and strategies for applying the PRMT6 molecule in the prevention and treatment of infectious diseases.

[0027] This application investigates the effects of PRMT6, a novel molecule with anti-infective activity, on the phagocytic and clearance effects of macrophages on bacteria in immune cells, and verifies the therapeutic and protective effects of applying the sequence of this molecule on bacterial infections in animals. Experiments demonstrate that: 1) PRTM6-deficient mice exhibit increased macrophage-mediated inflammatory responses in the pathogenesis of colitis; 2) PRTM6-deficient mice are more sensitive to endotoxins; 3) PRTM6-deficient macrophages produce increased IL-1β upon stimulation with LPS, Pam3CSK4, and poly(I:C).

[0028] This application targets different nucleotide and protein products of the PRMT6 gene to inhibit macrophage-mediated inflammatory responses and improve the survival rate of mice with inflammatory bowel disease. These inventions demonstrate that PRMT6-related products hold promise as an effective means of treating and preventing inflammatory bowel disease.

[0029] Example

[0030] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application. Those skilled in the art can make appropriate modifications and variations to the present application, and such modifications and variations are all within the scope of the present application.

[0031] Experimental methods not specifically described in the following examples can be performed using conventional methods in the art, such as those described in *Molecular Cloning: A Laboratory Manual* (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and can also be provided by commercial companies.

[0032] Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be used in the methods of this application. The preferred methods and materials described herein are for illustrative purposes only.

[0033] Unless otherwise specified, all reagents and equipment are commonly used in the field or are commercially available.

[0034] Example 1: PRTM6-deficient mice exhibit increased macrophage-mediated inflammatory responses in the pathogenesis of colitis.

[0035] 1. PRMT6-deficient mice and control mice

[0036] PRMT6-deficient mice were purchased from Southern Model Biotechnology. Their construction involved targeting S129 embryonic stem cells and implanting them into C57 pseudopregnant mice to obtain the F1 generation. These mice were then raised in an SPF (Special Purpose Free) environment to obtain PRMT6 knockout mice (i.e., PRMT6-deficient mice) and PRMT6-deficient mice. + / + Control mice (WT). Among them, PRMT6 protein expression was normal in WT controls.

[0037] WT and KO mice were sex- and weight-matched and used for 6-8 weeks, with mice weighing approximately 18-22g.

[0038] 2. Testing Methods

[0039] For seven consecutive days, PRMT6-deficient and control mice were fed a normal diet with 3% DSS added to distilled water. Survival rate, body weight, and health status were dynamically observed. Blood was collected from the eyeballs, and the serum production of inflammatory factors IL-1β, TNF-α, and IL-6 was detected using ELISA (purchased from R&D Company). Simultaneously, whole-cell RNA was collected, and the expression levels of cytokine RNA were detected using quantitative PCR (total RNA was extracted using the FAST200 Total RNA Isolation Kit (Cat#220011, Fastagen) and ReverTraAce). TM The qRT-PCR kit (FSQ-101, TOYOBO) was used for reverse transcription to cDNA, and the Hyperactive pG-MNaseCUT & RUNAssay Kit (HD101) was from Vayme (Nanjing, China).

[0040] HE staining histopathology: Mouse colon tissue was fixed in 10% neutral buffered formalin for 24-48 hours, dehydrated using a routine ethanol gradient, cleared with xylene, and embedded in paraffin. Sections (4-5 μm thick) were dewaxed and hydrated, then stained with HE: hematoxylin stained the nucleus for 5-10 minutes, separated by hydrochloric acid and ethanol, and then blued with running water; eosin stained the cytoplasm for 1-5 minutes; dehydrated using a gradient of ethanol, cleared with xylene, and mounted with neutral resin. Histopathological changes in the colon tissue were observed under a light microscope.

[0041] Immunofluorescence assay: Mouse colon paraffin sections were dewaxed, hydrated, antigen-retrieved, permeabilized, and blocked (with PBS containing 5% normal serum and 1% BSA). They were then incubated overnight at 4°C with a primary antibody against inflammatory cells (A300-929A, from Thermos Fisher Scientific (Waltham, USA)). After washing with PBS, they were incubated at room temperature in the dark for 1 hour with a fluorescein-labeled secondary antibody (Alexa Fluor® 488-labeled goat anti-rabbit IgG, 1:500). After washing with PBS, the nuclei were stained with DAPI, and the sections were mounted with anti-quenching mounting medium. The expression location and intensity of the target protein were observed under a fluorescence microscope / confocal microscope, and a semi-quantitative analysis was performed to assess the degree of inflammatory cell infiltration.

[0042] 3. Results and Discussion

[0043] Survival analysis results as follows Figure 1 As shown in A, PRMT6-deficient mice died earlier, and the survival rate of KO mice was significantly lower than that of WT mice.

[0044] KO mice showed greater weight loss (B), more severe enteritis symptoms (C), and shorter intestinal length (D).

[0045] Histopathology, such as Figure 1 As shown in E, PRMT6-deficient colitis model mice exhibit decreased histopathological scores and increased inflammatory cell infiltration.

[0046] The immunofluorescence results are shown in Figure G, indicating increased macrophage infiltration in the intestinal tissue of the KO mouse colitis model.

[0047] The results of ELISA and qPCR analysis of serum inflammatory factors IL-1β, TNF-α, and IL-6 production are as follows: Figure 1 The results, as shown in the HM, indicate that PRMT6-deficient mice produced increased levels of inflammatory factors IL-1β, TNF-α, and IL-6 after developing colitis, and the production of inflammatory factors in KO mice was significantly higher than that in WT mice.

[0048] This example demonstrates that PRTM6-deficient mice exhibit an increased inflammatory response during the pathogenesis of colitis.

[0049] Example 2: PRTM6-deficient mice are more sensitive to endotoxins

[0050] 1. PRMT6-deficient mice and control mice

[0051] Same as Example 1

[0052] 2. Testing Methods

[0053] PRMT6-deficient and control mice were injected with LPS (purchased from Merck (Darmstadt, Germany)) to dynamically observe the survival rate of the mice; histopathological examination was performed to detect lung tissue damage and inflammatory cell infiltration. Blood was collected from the eyeballs, and the serum production of inflammatory factors IL-1β, TNF-α, and IL-6 was detected by ELISA (purchased from R&D Company).

[0054] 3. Results and Discussion

[0055] The survival rate of LPS-induced sepsis model mice is as follows: Figure 2 As shown in A. The results showed that PRMT6-deficient mice died earlier, and the survival rate of KO mice was significantly lower than that of WT mice.

[0056] HE staining analysis results of mouse lungs are as follows Figure 2 As shown in Figure B, KO mice showed more severe lung damage and greater inflammatory cell infiltration.

[0057] The results of ELISA analysis of serum inflammatory factors IL-1β, TNF-α and IL-6 production are as follows: Figure 2 As shown in the CE diagram. The results showed that PRMT6-deficient mice produced increased levels of inflammatory factors IL-1β, TNF-α, and IL-6 after LPS induction, and the production of inflammatory factors in KO mice was significantly higher than that in WT mice.

[0058] This example demonstrates that PRMT6-deficient mice are more sensitive to endotoxins.

[0059] Example 3: Increased IL-1β production in PRTM6-deficient macrophages upon stimulation with LPS, Pam3CSK4, and poly(I:C)

[0060] 1. PRMT6-deficient mice and control mice

[0061] Same as Example 1

[0062] 2. Acquisition of macrophages

[0063] PRMT6-deficient mice were injected intraperitoneally with 2 ml of broth, and macrophages were obtained by rinsing the peritoneal cavity with culture medium four days later (same as Example 1).

[0064] 3. Testing Methods

[0065] Peritoneal macrophages (1×10⁶ cells / year) were treated with 100 ng / ml LPS (Merck, Darmstadt, Germany), Pam3CSK4, and poly(I:C) (Invivogen, Toulouse, France) (in 1640 medium) respectively. 5 Cells / ml RPMI 1640 medium).

[0066] Cell culture supernatant was collected, and cytokine production was measured by ELISA (using a kit purchased from R&D Company). Simultaneously, whole-cell RNA was collected, and the expression levels of cytokine RNA were detected by quantitative PCR. Pro-IL-1β protein levels were also detected by Western blotting.

[0067] 4. Results and Discussion

[0068] Inflammatory factors produced by peritoneal macrophages in PRMT6-deficient mice in response to LPS, Pam3CSK4, and poly(I:C) stimulation, such as... Figure 3 As shown in AE.

[0069] The results showed that peritoneal macrophages in PRMT6-deficient mice produced more inflammatory cytokine IL-1β after stimulation with LPS, Pam3CSK4, and poly(I:C).

[0070] The results indicate that PRMT6 deficiency increases the production of the inflammatory cytokine IL-1β in mouse-derived peritoneal macrophages.

[0071] Example 4: Macrophages overexpressing PRTM6 produced IL-1β attenuation upon stimulation with LPS, Pam3CSK4, and poly(I:C). few

[0072] 1. Plasmids and Cells

[0073] Wild-type PRTM6 plasmid (obtained by PCR cloning of full-length LAPF CDS into pCMV plasmid) and control plasmid (pCMV empty vector plasmid) were purchased from Invitrogen.

[0074] RAW264.7 cells (purchased from ATCC).

[0075] 2. Obtaining cells overexpressing PRTM6 and control cells

[0076] RAW264.7 cells were transfected with wild-type PRTM6 plasmid and control plasmid (using Jetpei reagent). After 48 hours, single clones were selected using 1500 ng / ml G418 to obtain stable RAW264.7 cells overexpressing PRTM6 and control cells (transfected with pCMV empty vector plasmid).

[0077] 3. Testing Methods

[0078] Macrophages (1 × 10⁶ cells) were treated with 100 ng / ml LPS (Merck, Darmstadt, Germany), Pam3CSK4, and poly(I:C) (Invivogen, Toulouse, France) in 1640 medium. 5 1 cell / ml RPMI 1640 medium).

[0079] Cell culture supernatant was collected, and whole-cell RNA was collected for quantitative PCR to detect the expression level of cytokine RNA. Pro-IL-1β protein level was detected by Western blotting.

[0080] 4. Results and Discussion

[0081] Macrophages overexpressing PRMT6 respond to LPS, Pam3CSK4, and poly(I:C) stimulation, resulting in the production of inflammatory factors such as... Figure 4 As shown in A and B.

[0082] The results showed that macrophages overexpressing PRMT6 produced less inflammatory cytokine IL-1β after stimulation with LPS, Pam3CSK4 and poly(I:C).

[0083] The results indicate that PRMT6 overexpression reduces the production of the macrophage inflammatory cytokine IL-1β.

[0084] Example 5: P50 inhibitors increase PRTM6 expression and alleviate symptoms in colitis mice

[0085] 1. PRMT6 promoter treatment in mice and control mice

[0086] C57 mice were purchased from Southern Model Organisms and raised in an SPF environment. During the modeling period, the PRMT6 promoter, kamebakaurin (KA) (selleck, E2582), was injected intraperitoneally every two days. The experimental group mice and control mice were matched for sex and weight and used for 6-8 weeks, with the mice weighing approximately 18-22g.

[0087] 2. Testing Methods

[0088] For seven consecutive days, experimental and control mice were fed a normal diet with 3% DSS added to distilled water. Survival rate, weight, and health status were dynamically observed. Blood was collected from the eyeballs, and the serum levels of inflammatory factors IL-1β, TNF-α, and IL-6 were detected using ELISA (purchased from R&D Company). Simultaneously, whole-cell RNA was collected, and the expression levels of cytokine RNA were detected using quantitative PCR (total RNA was extracted using the FAST200 Total RNA Isolation Kit (Cat#220011, Fastagen) and ReverTraAce). TM The qRT-PCR kit (FSQ-101, TOYOBO) was used for reverse transcription to cDNA, and the Hyperactive pG-MNaseCUT & RUNAssay Kit (HD101) was from Vazyme (Nanjing, China).

[0089] HE staining histopathology: Mouse colon tissue was fixed in 10% neutral buffered formalin for 24-48 hours, dehydrated using a routine ethanol gradient, cleared with xylene, and embedded in paraffin. Sections (4-5 μm thick) were dewaxed and hydrated, then stained with HE: hematoxylin stained the nucleus for 5-10 minutes, separated by hydrochloric acid and ethanol, and then blued with running water; eosin stained the cytoplasm for 1-5 minutes; dehydrated using a gradient of ethanol, cleared with xylene, and mounted with neutral resin. Histopathological changes in the colon tissue were observed under a light microscope.

[0090] Immunofluorescence assay: Mouse colon paraffin sections were dewaxed, hydrated, antigen-retrieved, permeabilized, and blocked (with PBS containing 5% normal serum and 1% BSA). They were then incubated overnight at 4°C with a primary antibody against inflammatory cells (A300-929A, from Thermos Fisher Scientific (Waltham, USA)). After washing with PBS, they were incubated at room temperature in the dark for 1 hour with a fluorescein-labeled secondary antibody (Alexa Fluor® 488-labeled goat anti-rabbit IgG, 1:500). After washing with PBS, the nuclei were stained with DAPI, and the sections were mounted with anti-quenching mounting medium. The expression location and intensity of the target protein were observed under a fluorescence microscope / confocal microscope, and a semi-quantitative analysis was performed to assess the degree of inflammatory cell infiltration.

[0091] 3. Results and Discussion

[0092] The expression analysis results of PRMT6 are as follows: Figure 5 As shown in A and B: KA promotes PRMT6 expression.

[0093] KA-treated mice experienced less weight loss (C), milder enteritis symptoms (D), and longer intestinal length (E).

[0094] Histopathology, such as Figure 5 As shown in F and G: KA-treated mice showed reduced histopathological scores and decreased inflammatory cell infiltration.

[0095] This example demonstrates that promoting PRMT6 expression via KA can alleviate colitis symptoms in mice.

[0096] The above are merely specific application examples of this application and do not constitute any limitation on the scope of protection of this application. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to list and describe all embodiments here. Any technical solutions similar to these obtained through equivalent transformations or substitutions fall within the scope of protection of this application.

[0097] Sequence information

Claims

1. Use of protein arginine methyltransferase 6 (PRMT6), its coding sequence or its promoter in the preparation of medicaments for the prevention and / or treatment of macrophage-mediated inflammatory responses.

2. The use as claimed in claim 1, wherein the inflammatory response is caused by a pathogen, a chemical substance, a physical factor, or a combination thereof.

3. The use as claimed in claim 1, wherein the inflammatory response includes inflammatory bowel disease, such as colitis.

4. Use of PRMT6, its coding sequence or its promoters in the preparation of medicaments for the prevention and / or treatment of infectious diseases or autoimmune diseases and their related conditions and / or symptoms.

5. The use as claimed in claim 4, wherein the associated symptoms and / or signs include one or more selected from the group consisting of: diarrhea, hematochezia, excessive production of inflammatory factors, inflammatory shock or death, inflammatory damage to organs, and multiple organ failure.

6. The use as claimed in claim 5, wherein the inflammatory factor comprises one or more selected from the group consisting of IL-1β, TNFα, and IL-6, preferably IL-1β; and / or The organs include digestive organs, such as the intestines and spleen.

7. A pharmaceutical composition comprising: (a) PRMT6, its coding sequence or its promoter; (b) A pharmaceutically or immunologically acceptable carrier or excipient; and (c) Other active ingredients used to prevent or treat macrophage-mediated inflammatory responses, infectious diseases or autoimmune diseases and their related conditions and / or symptoms.

8. The pharmaceutical composition of claim 7, wherein the other active ingredient comprises a biological agent and / or a small molecule drug, preferably comprising one or more selected from the group consisting of 5-aminosalicylic acid compounds, corticosteroids, and immunosuppressants.

9. The pharmaceutical composition of claim 7, wherein the PRMT6, its coding sequence or promoter accounts for 0.001-99.9 wt%, for example 1-95 wt%, preferably 5-90 wt%, more preferably 10-80 wt% of the total weight of the pharmaceutical composition.

10. The use as described in any one of claims 1-6 or the pharmaceutical composition as described in any one of claims 7-9, wherein: The PRMT6 is selected from: (i) The amino acid sequence of SEQ ID NO: 2 or 4; or (ii) An amino acid sequence that is at least 80% identical to SEQ ID NO: 2 or 4; and / or The PRMT6 encoding gene is selected from: (i) The nucleotide sequence of SEQ ID NO: 1 or 3; or (ii) A nucleotide sequence having at least 80% identity with SEQ ID NO: 1 or 3; and / or The PRMT6 promoter includes substances that increase PRMT6 protein levels or promote PRMT6 function, such as those selected from: PRMT6 expression vectors, naked DNA encoding the PRMT6 sequence, liposome-encapsulated DNA encoding the PRMT6 sequence, LAFP precursor proteins or conjugates or complexes that can be converted into PRMT6 in vivo, PRMT6 agonists, or combinations thereof.

Images