WO2024228570A1 - Découverte d'une cible pour inhiber la fibrose dans une maladie intestinale inflammatoire - Google Patents

Découverte d'une cible pour inhiber la fibrose dans une maladie intestinale inflammatoire Download PDF

Info

Publication number
WO2024228570A1
WO2024228570A1 PCT/KR2024/005979 KR2024005979W WO2024228570A1 WO 2024228570 A1 WO2024228570 A1 WO 2024228570A1 KR 2024005979 W KR2024005979 W KR 2024005979W WO 2024228570 A1 WO2024228570 A1 WO 2024228570A1
Authority
WO
WIPO (PCT)
Prior art keywords
nox4
colitis
dss
fibrosis
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2024/005979
Other languages
English (en)
Korean (ko)
Inventor
남기택
이유라
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University Industry Foundation UIF of Yonsei University
Original Assignee
University Industry Foundation UIF of Yonsei University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Industry Foundation UIF of Yonsei University filed Critical University Industry Foundation UIF of Yonsei University
Publication of WO2024228570A1 publication Critical patent/WO2024228570A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the discovery of a target for suppressing fibrosis in inflammatory bowel disease and the prediction of the progression of fibrosis in inflammatory bowel disease using the same.
  • Ulcerative colitis is a common form of inflammatory bowel disease (IBD) caused by long-term inflammatory damage and ulceration of the colonic mucosa. Persistent inflammation induced by commensal bacteria affects the intestinal epithelial barrier and is considered a major cause of UC development.
  • IBD inflammatory bowel disease
  • current treatments are ineffective for most UC patients and are associated with adverse effects such as tissue dysfunction, fibrostenosis, and malignant transformation.
  • UC-related carcinogenesis is mainly caused by excessive reactive oxygen species (ROS) generated by inflammation in damaged tissues, and the resulting damage to redox balance induces oxidative stress due to genetic mutations and molecular damage such as lipids, DNA, and proteins. This ultimately transforms colon cells and causes colon cancer. Given the poor prognosis of UC-related colon cancer, it is urgent to identify new therapeutic targets.
  • ROS reactive oxygen species
  • NOX nicotinamide adenine dinucleotide phosphate
  • NADPH nicotinamide adenine dinucleotide phosphate
  • NOX1 and DUOX2 are abundantly expressed in the intestinal epithelium as major sources of ROS in the intestinal mucosa, and their mutations are associated with the risk of adult IBD.
  • NOX1 contributes to intestinal homeostasis by regulating mucus cell differentiation, migration, microbial defense, and mediating mucus repair after injury, while DUOX2 is activated by pathogen-derived uracil to generate DUOX-dependent ROS and eliminate gut microbes caused by intestinal infections.
  • NOX4 acts as an oxygen sensor and catalyzes the reduction of molecular oxygen to various reactive oxygen species (ROS), which have different functions depending on the organ. NOX4 appears to be associated with the development of ureteral obstruction and pulmonary hypertension, and induces activation of FLT3 (fms-like tyrosine kinase 3) signaling and cellular responses to stimulation in the disease.
  • ROS reactive oxygen species
  • NOX4 Several recent studies have revealed a role for NOX4 in modulating inflammatory-mediated pathophysiology in the colon. NOX4 generates H 2 O 2 and is predominantly expressed in vascular smooth muscle cells, endothelial cells, and fibroblasts. We found that Nox4 transcripts are detected at low levels in normal colon, but NOX4 expression is upregulated in drug-resistant patients with fibrostenosis in UC or Crohn's disease (CD) and in a dextran sulfate sodium (DSS)-induced murine colitis model. Previously, NOX4 was not associated with the development of fibrotic features in mouse colitis models, and the detailed molecular mechanisms underlying fibrosis in UC have not been elucidated.
  • NOX4 contributes to intestinal fibrotic damage in ulcerative colitis and that TGF- ⁇ signaling is required to induce fibrosis development.
  • TGF- ⁇ signaling is required to induce fibrosis development.
  • NOX4-deficient mice Nox4 -/-
  • DSS-induced colitis investigated the effect of Nox4 on the response to intestinal damage. This study is expected to be greatly utilized in predicting whether fibrosis progresses in inflammatory bowel disease and suppressing it.
  • NOX4 contributes to intestinal fibrotic damage in ulcerative colitis and that TGF- ⁇ signaling is required to induce fibrosis development.
  • TGF- ⁇ signaling is required to induce fibrosis development.
  • the purpose of the present invention is to elucidate the role of TGF- ⁇ signaling in contributing to the intestinal fibrotic damage of colitis and inducing the development of fibrosis in ulcerative colitis.
  • Another object of the present invention is to provide a composition for predicting whether fibrosis progresses in colitis.
  • Another object of the present invention is to provide a kit for predicting whether fibrosis progresses in colitis.
  • Another object of the present invention is to provide a method for predicting whether fibrosis progresses in colitis.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating fibrosis in colitis.
  • Nox4 is considered to be closely related to tissue fibrosis, but its specific role in the progression of fibrosis has been controversial. Nox4-induced ROS appear to induce tissue fibrosis by promoting apoptosis and mesenchymal cell differentiation and activation. Nox4 has been reported to play an important role in the mouse lung, inducing alveolar cell death by myofibroblast activation and subsequent bleomycin-induced fibrosis, and attenuating CCl4-induced liver injury, hepatocyte apoptosis, and liver fibrosis (Nat Med 2009;15:1077-81.).
  • Nox4 contributed to the prevention of renal fibrosis in the kidneys of mice with chronic kidney injury by inhibiting tubular cell apoptosis and oxidative stress (J Am Soc Nephrol 2012;23:1967-76.).
  • Nox4 inhibits ROS production by reducing Hif1a expression and antioxidant molecules in the Keap1-Nrf pathway, suggesting that Nox4 may positively regulate antioxidant activity in the kidney.
  • TGF- ⁇ signaling pathway has been extensively linked to tissue physiology, including collagen synthesis and Treg cell lineage determination.
  • loss of Nox4 altered the subset of T cells stimulated by persistently activated TGF- ⁇ in both normal and inflamed mouse colons.
  • TGF- ⁇ signaling could directly induce differentiation of Foxp3 + Tregs and RORgT + Th17 cells from naive CD4 + T cells.
  • Nox4 -/- mice had a marked increase in the infiltration of Foxp3 + Treg cells.
  • Tregs are specifically involved in immune suppression against commensal bacteria in the gut.
  • Tregs The infiltration of Foxp3 + Tregs, together with increased ROS in Nox4 -/- colons, induced TGF- ⁇ activation, expression of proinflammatory cytokines (Tnf and Il1 ⁇ ), and infiltration of F4/80 + macrophages. Therefore, the increase in Tregs is considered not only due to TGF- ⁇ activation but also represents a defense mechanism that regulates the inflammatory immune response in the colon of Nox4 -/- mice.
  • Th17 cells are more abundant in patients with active IBD and play a causative role in IBD-related colorectal cancer. Colorectal cancer is usually densely infiltrated with proinflammatory cytokine-producing cells. Among tumor-infiltrating immune cells, T cells are transformed into a functional population that secretes large amounts of Th17-inducing cytokines (IL-17A, IL-12, IL-22), TNF- ⁇ , and IL-6. This population promotes colon tumor formation and survival through STAT3/NF- ⁇ B activation. Our findings suggest that Th17 cells and associated cytokines can enhance the survival and growth of colorectal cancer in Nox4 -/- mice.
  • Th17-inducing cytokines IL-17A, IL-12, IL-22
  • Nox1 and Duox2 are expressed in the intestinal epithelium and are major sources of ROS in the gastrointestinal tract. Deficiency of these genes is associated with increased disease severity in colitis.
  • Conditional deletion of Nox4 in fibroblasts and enterocytes results in a higher tumor burden, especially in endothelial cells, and fibroblast-specific deletion of Nox4 resulted in faster tumor development than epithelial cell-specific deletion in Nox4 mice.
  • Nox4 is expressed in non-epithelial cell populations, suggesting that Nox4 may determine the severity of colonic damage by regulating ROS-induced TGF- ⁇ signaling in colonic stromal cells.
  • Nox4 In conclusion, in this study, we have shown that genetic deletion of Nox4 leads to more severe fibrosing colitis progression and subsequent failure of recovery. Loss of Nox4 increased oxidative stress in colonic tissues and subsequently permanently activated canonical TGF- ⁇ signaling. In addition, it affected the differentiation and proliferation of T cell lineages (Treg and Th17 cells) mediated by TGF- ⁇ , and the progression of fibrosis in colitis. Our findings on the role of Nox4 in murine colitis suggest that modulation of redox signaling may be an important target for successful treatment of IBD patients.
  • the present invention comprises a preparation capable of measuring the expression level of Nox4 (NADPH Oxidase 4) protein, or a gene encoding the same, and capable of measuring the expression level of any one or more proteins selected from the group consisting of CD3e (Cluster of Differentiation 3 epsilon), Mucl1 (Mucin Like 1), Vegf (vascular endothelial growth factor), Ano5 (Anoctamin 5), Tnfrsf9 (TNF Receptor Superfamily Member 9), Itg2a (Integrin ⁇ 2), Nlrp3 (NLR family pyrin domain containing 3), Tnfrsf4 (TNF Receptor Superfamily Member 4), Fabp7 (Fatty Acid Binding Protein 7), CD79b (Cluster of Differentiation 79B), Ptgs2 (Prostaglandin-endoperoxide synthase 2), and Caprin2 (Caprin Family Member 2), or a gene encoding the same, and capable of
  • the colitis may be ulcerative colitis.
  • the biological sample is selected from the group consisting of tissue, cell, cell extract, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, It may be any one or more selected from the group consisting of synovial fluid, joint aspirate, organ secretions, and cerebrospinal fluid.
  • the preparation for measuring the expression level of the protein may include at least one selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNAs), and aptamers that specifically bind to the protein.
  • the antibody refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction.
  • the antibody means an antibody that specifically binds to a protein that functions as the biomarker.
  • the antibody of the present invention includes all of polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibody can be easily produced using techniques well known in the art.
  • the polyclonal antibody can be produced by a method well known in the art including a process of injecting an antigen of the biomarker protein into an animal and collecting blood from the animal to obtain serum containing the antibody.
  • Such polyclonal antibodies can be produced from any animal, such as a goat, a rabbit, a sheep, a monkey, a horse, a pig, a cow, or a dog.
  • monoclonal antibodies can be produced using the hybridoma method widely known in the art (European Journal of Immunology, 6, 511-519.) or the phage antibody library technology (Mol. Biol., 222:58, 1-597, 1991).
  • the antibodies produced by the above methods can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography.
  • the antibody of the present invention includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of an antibody molecule.
  • a functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.
  • the PNA Peptide Nucleic Acid
  • PNA Peptide Nucleic Acid
  • DNA has a phosphate-ribose sugar backbone
  • PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases the binding force and stability to DNA or RNA, and is thus used in molecular biology, diagnostic analysis, and antisense therapy.
  • PNA is disclosed in detail in the literature [Science 254 (5037): 1497-1500].
  • the aptamer is an oligonucleic acid or peptide molecule, and the general contents of the aptamer are disclosed in detail in the literature [Proc Natl Acad Sci USA. 95(24): 142727(1998)].
  • the preparation for measuring the expression level of the gene may include at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to the gene.
  • the primer is a fragment that recognizes a target gene sequence, and includes a pair of forward and reverse primers, but is preferably a pair of primers that provides an analysis result with specificity and sensitivity.
  • the nucleic acid sequence of the primer is a sequence that does not match a non-target sequence existing in the sample, and thus amplifies only the target gene sequence containing a complementary primer binding site and does not cause non-specific amplification, high specificity can be provided.
  • the probe means a substance that can specifically bind to a target substance to be detected in the sample, and means a substance that can specifically confirm the presence of the target substance in the sample through the binding.
  • the type of the probe is not limited to a substance commonly used in the art, but is preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA.
  • the probe includes a biomaterial derived from or similar to a living organism or manufactured in vitro, for example, an enzyme, a protein, an antibody, a microorganism, an animal or plant cell and organ, a nerve cell, DNA, and RNA.
  • DNA includes cDNA, genomic DNA, and oligonucleotides
  • RNA includes genomic RNA, mRNA, and oligonucleotides
  • proteins include antibodies, antigens, enzymes, peptides, and the like.
  • the LNA (Locked nucleic acids) in the present invention means a nucleic acid analog including a 2'-O, 4'-C methylene bridge (Gene Therapy (2006) 13, 496.502).
  • LNA nucleosides include general nucleic acid bases of DNA and RNA, and can form base pairs according to the Watson-Crick base pairing rule. However, due to the 'locking' of the molecule due to the methylene bridge, LNA cannot form an ideal shape in Watson-Crick binding.
  • the antisense means an oligomer having a sequence of nucleotide bases and a backbone between subunits that allows the antisense oligomer to hybridize with a target sequence in RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA:oligomer heteroduplex within the target sequence.
  • the oligomer may have exact sequence complementarity or approximate complementarity to the target sequence.
  • the present invention comprises a preparation capable of measuring the expression level of Nox4 (NADPH Oxidase 4) protein, or a gene encoding the same, and capable of measuring the expression level of any one or more proteins selected from the group consisting of CD3e (Cluster of Differentiation 3 epsilon), Mucl1 (Mucin Like 1), Vegf (vascular endothelial growth factor), Ano5 (Anoctamin 5), Tnfrsf9 (TNF Receptor Superfamily Member 9), Itg2a (Integrin ⁇ 2), Nlrp3 (NLR family pyrin domain containing 3), Tnfrsf4 (TNF Receptor Superfamily Member 4), Fabp7 (Fatty Acid Binding Protein 7), CD79b (Cluster of Differentiation 79B), Ptgs2 (Prostaglandin-endoperoxide synthase 2), and Caprin2 (Caprin Family Member 2), or a gene encoding the same, and capable of
  • the kit may be a RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit or an MRM (Multiple reaction monitoring) kit.
  • the kit of the present invention may further include one or more kinds of other component compositions, solutions or devices suitable for the analysis method.
  • the kit of the present invention may further include essential elements necessary for performing a reverse transcription polymerase reaction.
  • the reverse transcription polymerase reaction kit includes a primer pair specific for a gene encoding a marker protein.
  • the primers are nucleotides having a sequence specific for the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp.
  • kits may include a primer specific for the nucleic acid sequence of a control gene.
  • Other reverse transcription polymerase reaction kits may include a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, and the like.
  • the kit of the present invention may include essential elements necessary for performing a DNA chip.
  • the DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, agents, enzymes, and the like for producing a fluorescently labeled probe.
  • the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.
  • the kit of the present invention may include essential elements necessary for performing an ELISA.
  • the ELISA kit includes an antibody specific for the protein. Antibodies are antibodies that have high specificity and affinity for the marker protein and little cross-reactivity to other proteins, and are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies.
  • the ELISA kit may include antibodies specific for a control protein.
  • the ELISA kit may include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to the antibody), and their substrates or other substances that can bind to the antibody.
  • the present invention provides a method for treating colitis, comprising the steps of: (a) confirming an increase in the expression of Nox4 (NADPH Oxidase 4) protein, or a gene encoding the same, in a biological sample isolated from a subject with colitis; And, (b) a step of confirming an increase or decrease in the expression of at least one protein selected from the group consisting of CD3e (Cluster of Differentiation 3 epsilon), Mucl1 (Mucin Like 1), Vegf (vascular endothelial growth factor), Ano5 (Anoctamin 5), Tnfrsf9 (TNF Receptor Superfamily Member 9), Itg2a (Integrin ⁇ 2), Nlrp3 (NLR family pyrin domain containing 3), Tnfrsf4 (TNF Receptor Superfamily Member 4), Fabp7 (Fatty Acid Binding Protein 7), CD79b (Cluster of Differentiation 79B), P
  • the colitis may be ulcerative colitis.
  • the biological sample is selected from the group consisting of tissue, cell, cell extract, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, It may be any one or more selected from the group consisting of synovial fluid, joint aspirate, organ secretions, and cerebrospinal fluid.
  • the method is a method of predicting that there is a high possibility that fibrosis will progress in the colitis when the expression of any one or more proteins selected from the group consisting of CD3e, Mucl1, Vegf, Ano5, Tnfrsf9, Itg2a, and Nlrp3, or a gene encoding the same, is increased.
  • the method is a method for predicting that there is a high possibility that fibrosis will progress in the colitis when the expression of any one or more proteins selected from the group consisting of Tnfrsf4, Fabp47, CD79b, Ptgs2, and Caprin2, or a gene encoding the same, is reduced.
  • the increase or decrease in expression of the protein or gene may be more than twice that of the normal control group.
  • the method is a method in which an increase in the expression of Nox4 (NADPH Oxidase 4) protein, or a gene encoding the same, is regulated by TGF- ⁇ (Transforming growth factor-beta).
  • Nox4 NADPH Oxidase 4
  • TGF- ⁇ Transforming growth factor-beta
  • the method may further include a step of (c) confirming an increase in F4/80 + M1 macrophages, CD127 + lymphocytes, regulatory T cells, or ROR ⁇ T + Th17 cells (T helper 17 cells).
  • a step of (c) confirming an increase in F4/80 + M1 macrophages, CD127 + lymphocytes, regulatory T cells, or ROR ⁇ T + Th17 cells T helper 17 cells.
  • the method may further include a step of (c) confirming a decrease in CD163 + M2 macrophages.
  • the CD163 + M2 macrophages are decreased compared to the normal control group, it is a method of predicting that there is a high possibility that fibrosis will progress in the colitis.
  • the present invention provides a pharmaceutical composition for preventing or treating fibrosis in colitis, comprising an expression inhibitor of Nox4 (NADPH Oxidase 4) protein, or a gene encoding the same, and further comprising an expression inhibitor of at least one protein selected from the group consisting of CD3e (Cluster of Differentiation 3 epsilon), Mucl1 (Mucin Like 1), Vegf (vascular endothelial growth factor), Ano5 (Anoctamin 5), Tnfrsf9 (TNF Receptor Superfamily Member 9), Itg2a (Integrin ⁇ 2), and Nlrp3 (NLR family pyrin domain containing 3), or a gene encoding the same.
  • CD3e Cluster of Differentiation 3 epsilon
  • Mucl1 Mocl1
  • Vegf vascular endothelial growth factor
  • Ano5 Anoctamin 5
  • Tnfrsf9 TNF Receptor Superfamily Member 9
  • the colitis may be ulcerative colitis.
  • the present invention provides a pharmaceutical composition for preventing or treating fibrosis in colitis, comprising an inhibitor of expression of Nox4 (NADPH Oxidase 4) protein, or a gene encoding the same, and further comprising an activator of expression of at least one protein selected from the group consisting of Tnfrsf4 (TNF Receptor Superfamily Member 4), Fabp7 (Fatty Acid Binding Protein 7), CD79b (Cluster of Differentiation 79B), Ptgs2 (Prostaglandin-endoperoxide synthase 2), and Caprin2 (Caprin Family Member 2), or a gene encoding the same.
  • Tnfrsf4 TNF Receptor Superfamily Member 4
  • Fabp7 Fatty Acid Binding Protein 7
  • CD79b Cluster of Differentiation 79B
  • Ptgs2 Prostaglandin-endoperoxide synthase 2
  • Caprin2 Caprin Family Member 2
  • the colitis may be ulcerative colitis.
  • the term prevention means suppressing the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.
  • treatment means all actions that improve symptoms caused by the target disease or benefit the individual by using the effective ingredient of the present invention, and means an attempt to obtain useful results or desirable results including clinical results.
  • Useful or desirable clinical results may include, but are not necessarily limited to, alleviation or improvement of one or more symptoms or conditions, reduction of the extent of the disease, stabilization of the disease state, inhibition of disease occurrence, inhibition of disease spread, delay or slowing of disease progression, delay or slowing of disease onset, improvement or alleviation of the disease state, and reduction (partial or complete).
  • treatment may mean prolonging the survival of a patient beyond what would be expected in the absence of treatment.
  • treatment may mean inhibition of disease progression, temporary slowing of disease progression, and more preferably relates to permanently stopping disease progression.
  • treatment may mean inhibition of disease progression, temporary slowing of disease progression, and more preferably relates to permanently stopping disease progression.
  • results may be unbeneficial or undesirable.
  • composition of the present invention can be a composition for treating these diseases by itself, or can be administered together with other anticancer agents and applied as a therapeutic adjuvant for the diseases.
  • treatment or “therapeutic agent” in this specification includes the meaning of “therapeutic adjuvant” or “therapeutic adjuvant.”
  • the expression inhibitor of any one or more proteins selected from the group consisting of Nox4, CD3e, Mucl1, Vegf, Ano5, Tnfrsf9, Itg2a, and Nlrp3 may include an antibody specific for the target protein, or an antigen-binding fragment thereof.
  • Inhibition of protein activity here means inhibiting the activity of the target protein with an antibody specific for the protein or an antigen-binding fragment thereof.
  • the antibody is a term known in the art and means a specific protein molecule directed against an antigenic site.
  • Such antibodies can be produced by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene, and then producing the obtained protein by a conventional method.
  • the form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody, or a part thereof having antigen binding property are also included in the antibody of the present invention, and all immunoglobulin antibodies are included.
  • the antibody of the present invention also includes special antibodies such as humanized antibodies.
  • the antibody of the present invention includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of an antibody molecule.
  • the functional fragment of an antibody molecule means a fragment having at least an antigen binding function, and includes Fab, F(ab'), F(ab') 2, and Fv.
  • the expression inhibitor of any one or more genes selected from the group consisting of Nox4, CD3e, Mucl1, Vegf, Ano5, Tnfrsf9, Itg2a, and Nlrp3 may be any one or more selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and micro RNA specific for the target gene.
  • antisense oligonucleotides are short lengths of DNA synthetic strands (or DNA analogs) that are antisense (or complementary) to a specific DNA or RNA target, and are used to achieve gene-specific inhibition in vitro as well as in vivo.
  • Antisense oligonucleotides have been proposed to block the expression of proteins encoded by DNA or RNA targets by binding to the target and stopping expression at the stage of transcription, translation or splicing.
  • Antisense oligonucleotides have been successfully used in cell culture and animal models of disease. Other modifications of antisense oligonucleotides to make the oligonucleotides more stable and resistant to degradation are known and understood by those skilled in the art.
  • the antisense oligonucleotides used herein include double-stranded or single-stranded DNA, double-stranded or single-stranded RNA, DNA/RNA hybrids, DNA and RNA analogs, and oligonucleotides having base, sugar or backbone modifications.
  • the oligonucleotides are modified by methods known in the art to increase stability and increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications of the oligonucleotide backbone, modifications of the sugar moiety, or modifications of the base, which are known in the art.
  • the siRNA small interfering RNA
  • the siRNA molecule is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and is used as an efficient gene knockdown method or gene therapy method because it can suppress the expression of a target gene.
  • the siRNA molecule may have a structure in which the sense strand and the antisense strand are positioned opposite each other to form a double-stranded structure, or a single-stranded structure having self-complementary sense and antisense strands.
  • the siRNA is not limited to a double-stranded RNA portion in which RNA is paired with another RNA being completely paired, and may include a portion that does not pair due to a mismatch (corresponding bases are not complementary), a bulge (there is no corresponding base in one chain), etc.
  • the siRNA terminal structure can be either a blunt end or a cohesive end as long as it can suppress the expression of the target gene by RNA interference (RNAi) effect.
  • the cohesive end structure can be either a 3'-end protruding structure or a 5'-end protruding structure.
  • the siRNA molecule is not limited thereto, but the total length can be 15 to 30 bases, preferably 19 to 21 bases.
  • shRNA short hairpin RNA
  • shRNA short hairpin RNA
  • the oligo DNA is cloned into a plasmid vector or the shRNA is expressed by inserting it into a retrovirus such as lentivirus and adenovirus, an shRNA having a hairpin structure with a loop is created and converted into siRNA by dicer in the cell to exhibit an RNAi effect.
  • the above microRNA regulates various biological processes such as development, differentiation, proliferation, preservation, and apoptosis.
  • MicroRNA generally regulates the expression of a gene encoding a target mRNA by destabilizing the target mRNA or interfering with its translation.
  • Regulatory sequences useful for the expression construct/vector having the antisense oligonucleotide, siRNA, shRNA or microRNA e.g., a constitutive promoter, an inducible promoter, a tissue-specific promoter or a combination thereof
  • the expression inhibitor of the gene can be any substance that inhibits the expression of the gene in addition to the antisense oligonucleotide, siRNA, shRNA or microRNA.
  • compositions of the present invention are not limited thereto, but may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, each according to a conventional method.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. for oral administration, and may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc.
  • formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above.
  • for oral administration it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.
  • for injections it can be manufactured in the form of unit dosage ampoules or multiple dosage forms.
  • it can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.
  • examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil.
  • fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.
  • administration in the present specification means directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject, and includes introducing the composition of the present invention to a patient by any appropriate method, and the administration route of the composition of the present invention may be administered through any general route as long as it can reach the target tissue.
  • Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration, intracavity administration, intraperitoneal administration, and intrathecal administration may be performed, but is not limited thereto.
  • the effective amount in the present invention may be adjusted according to various factors including the type of disease, the severity of the disease, the type and content of the effective ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health condition, sex, and diet, administration time, administration route, and secretion rate of the composition, treatment period, and concurrently used drugs.
  • the pharmaceutical composition for treatment can be administered into the body in an amount of 50 ml to 500 ml at a time, and in the case of a compound, it can be administered in a dose of 0.1 ng/kg to 10 mg/kg, and in the case of a monoclonal antibody, it can be administered in a dose of 0.1 ng/kg to 10 mg/kg.
  • the administration interval can be 1 to 12 times a day, and in the case of administration 12 times a day, it can be administered once every 2 hours.
  • the pharmaceutical composition of the present invention can be administered alone or together with other treatments known in the art, such as chemotherapy, radiation, and surgery, for the treatment of the intended cancer.
  • the pharmaceutical composition of the present invention may be administered in combination with other treatments designed to enhance an immune response, for example, adjuvants or cytokines (or nucleic acids encoding cytokines) as are well known in the art.
  • Other standard delivery methods such as biolistic delivery or ex vivo treatment, may also be used.
  • antigen-presenting cells for example, antigen-presenting cells (APCs), dendritic cells, peripheral blood mononuclear cells, or bone marrow cells may be obtained from a patient or a suitable donor, activated ex vivo with the pharmaceutical composition, and then administered to the patient.
  • the term therapeutically effective amount at this time means the content of the composition containing a pharmacological ingredient in the composition in an amount sufficient to provide a therapeutic or prophylactic effect to a subject to whom the pharmaceutical composition of the present invention is to be administered, and thus includes a prophylactically effective amount.
  • the researchers have elucidated that genetic deletion of Nox4 leads to the progression of more severe fibrosing colitis and subsequent failure of recovery.
  • Loss of Nox4 increased oxidative stress in colonic tissue and subsequently permanently activated canonical TGF- ⁇ signaling.
  • it affected the differentiation and proliferation of T cell lineages (Treg and Th17 cells) mediated by TGF- ⁇ , and the progression of fibrosis in colitis. Therefore, the present invention is expected to be greatly utilized in predicting whether fibrosis progresses in inflammatory bowel disease and suppressing it.
  • Figure 1 shows the generation of Nox4 -/- mice.
  • Figures 1a and 1b are schematic representations of Nox2 or Nox4 deletions using CRISPR/Cas9 technology. The generated heterozygous and homozygous mice were genotyped by PCR using specific Nox2 or Nox4 deletion regions.
  • Figure 1c left shows the ISH results for mNox4 in untreated and 2.5% DSS-treated colon sections, and Figure 1c right shows the immunohistochemical results for Nox2 in untreated and 2.5% DSS-treated colon sections.
  • Figure 1d shows the Sirius-Red staining results showing fibrotic areas in red. The graph shows the numerical measurement of fibrotic areas using Image J.
  • Figures 1e and 1f show the levels of intracellular reactive oxygen species (ROS) assessed using DCFH-DA staining in untreated or treated groups. Data are presented as mean ⁇ SD, and significance is *P ⁇ 0.5, **P ⁇ 0.01 compared to wild type (WT).
  • ROS reactive oxygen species
  • Figure 2 shows that Nox4 has a protective function in experimental colitis induced by dextran sodium sulfate (DSS).
  • Figure 2a shows the experimental plan for establishing a DSS-induced murine colitis model.
  • Figure 2d shows the macroscopic observations of the colons of WT and Nox4 -/- mice with DSS-induced colitis sacrificed on day 14. The colon length was assessed after euthanasia of the mice.
  • Figure 2e shows representative hematoxylin and eosin staining images of the colon tissues of WT and Nox4 -/- mice with DSS-induced colitis.
  • Figure 2f shows the colitis score for pathological evaluation of DSS-induced colitis in WT and Nox4 -/- mice. The inflammation severity was scored as none (0), mucosal inflammation (1), submucosal inflammation (2), and transmural inflammation (3) according to the degree of inflammation.
  • Figure 2g shows the disease activity index, which consists of daily measured body weight loss, stool consistency, and rectal bleeding assessment. Data are expressed as mean ⁇ SD, and the significance is *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.005, ****P ⁇ 0.001 compared to wild type (WT).
  • Figure 3 shows that Nox4 is involved in intestinal fibrosis and immune-mediated tissue regeneration.
  • Figure 3a shows the results of immunohistochemical (IHC) staining of F4/80 (top), indicating M1 macrophages, CD164 (middle), indicating M2 macrophages, and CD127 (bottom), indicating lymphoid cells, in colon sections of wild-type (WT) and Nox4 -/- mice treated with dextran sulfate sodium (DSS).
  • Figure 3b shows the results of immunohistochemical (IHC) staining for Ki-67, indicating proliferating cells. IHC quantification of the 2.5% DSS treatment group was evaluated using ImageJ software, and the results are displayed as bar graphs on the right side of the images.
  • Figure 3c shows the results of Sirius Red staining (top), showing the fibrotic area in red, and immunolabeling using Tenascin-C antibody, indicating fibrosis of colonic tissue (bottom). Each positive staining area was measured by ImageJ, and the results are displayed as a bar graph next to each image.
  • Fig. 3d shows the RT-qPCR results of the inflammation-related genes Tnf and Il1b, and Fig.
  • 3e shows the RT-qPCR results of the fibrosis-related genes Col1a1, Col3a1, and Tnc.
  • the data are expressed as the mean ⁇ SD, and the significance is *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.005, ****P ⁇ 0.001 compared to the wild type (WT).
  • Figure 4 shows the results of immune cell infiltration in the colons of wild-type (WT) and Nox4 -/- mice.
  • Figure 4a is the result of immunohistochemical (IHC) staining for F4/80 (top) representing M1 macrophages, CD164 (middle) representing M2 macrophages, and CD127 (bottom) representing lymphocyte cells.
  • Figure 5 shows that loss of Nox4 leads to collagen synthesis and infiltration of regulatory T cell (Treg) lineages.
  • Figure 5b is a 2-dimensional and 3-dimensional principal component analysis (PCA) plot for the molecular signatures of colon tissue from WT and Nox4 -/- groups untreated or treated with DSS.
  • Figure 5c is a volcano plot of genes that are down-regulated (blue) or up-regulated (red) with a difference of more than 2-fold in expression level in Nox4 -/- compared to the WT group.
  • PCA principal component analysis
  • Figure 5d is a gene ontology (GO) biological process (BP) analysis of DEGs up-regulated in Nox4 -/- mice.
  • Figure 5e is the KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis result of Nox4 -/- DEG. The top 14 pathways are identified and the color represents statistical significance.
  • Figure 5f is the T cell subpopulation evaluated by CIBERSORT based on WT and Nox4 -/- DEG.
  • Figure 5g is the CIBERSORT analysis result of WT and Nox4 -/- colon tissue. The relative proportions of 11 different immune cell types were isolated from RNA sequencing data using CIBERSORT.
  • Figure 5h is the flow cytometry analysis result of distal colon tissue isolated from WT or Nox4 -/- mice, showing the relative number of viable cells coupled to CD4 + Foxp3 + T cells (left, histogram) and the number of CD4 + Foxp3 + T cells (right, bar graph).
  • Figure 5i is the gene set enrichment analysis result based on WT or Nox4 -/- DEG. The TGF- ⁇ gene set is enriched for genes upregulated in the colon of Nox4 -/- mice.
  • Figure 6 shows that Nox4 inhibits activation of TGF- ⁇ signaling in vivo.
  • Figure 6a shows the general TGF- ⁇ signaling pathway system.
  • black dots represent WT mice not treated with DSS
  • red dots represent Nox4 -/- mice.
  • Figure 6c shows the levels of active TGF- ⁇ in WT (black) or Nox4 -/- (red) colon tissue lysates measured by enzyme-linked immunosorbent assay.
  • Figure 6d shows the results of immunoblotting for TGF- ⁇ and TGFbR1 protein levels in WT or Nox4 -/- colon tissue extracts.
  • ⁇ -Actin was used as an internal control, and the band sizes were quantified by ImageJ.
  • Figure 6e shows the immunoblotting results of pSmad2/3, Smad2/3, and Smad4 protein levels in cytoplasmic and nuclear fraction extracts of colon tissues of WT and Nox4 -/- mice. GAPDH and HDAC were used as cytoplasmic and nuclear regulatory protein controls, respectively, and the band sizes were quantified by ImageJ.
  • Figure 6f shows TGF- ⁇ immunofluorescence staining results of normal and inflamed colon sections from WT or Nox4 -/- mice
  • Figure 6g shows immunofluorescence staining results of TGF- ⁇ /CD11c/DAPI. Data are expressed as mean ⁇ SD, and the significance is *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.005, ****P ⁇ 0.001 compared with wild type (WT).
  • Figure 7 shows that TGF- ⁇ signaling and its downstream molecules are upregulated in Nox4 -/- mice with dextran sodium sulfate (DSS)-induced colitis.
  • Figure 7a is a local Fisher discriminant analysis plot for principal component analysis. The two-dimensional scores are readily distinguished between W/DSS WT (green) and W/DSS Nox4 -/- (red) groups, and the shared regions represent 95% confidence intervals.
  • Figure 7b is the result of applying RNA sequencing to total colon cells sorted from W/DSS WT or W/DSS Nox4 -/- mice. A heatmap of the RNA sequencing data shows upregulated (purple) and downregulated (yellow) genes.
  • Figure 7c is a volcano plot identifying significantly upregulated genes in W/DSS WT (white) and W/DSS Nox4 -/- (red) mice.
  • Figures 7d and 7e show CIBERSORT analysis of WT and Nox4 -/- mouse colon tissues. The relative proportions of 11 different immune cell types (Fig. 7d) and 7 T cell lineage cell types (Fig. 7e) were separated from RNA sequencing data using CIBERSORT.
  • Figure 7f is a bubble plot showing significantly enriched KEGG pathways in differentially expressed genes (DEGs) in W/DSS WT and W/DSS Nox4 -/- groups. The bubble color represents the p-value and the bubble size represents the expression level.
  • DEGs differentially expressed genes
  • Figure 7g is a heatmap for TGF- ⁇ -related gene expression of W/DSS Nox4 -/- DEGs.
  • Figure 7h is immunoblotting results of TGF- ⁇ and TGFbR1 protein levels from W/DSS WT or W/DSS Nox4 -/- distal colon tissue extracts. ⁇ -Actin was used as an internal control, and the band sizes were quantified by ImageJ.
  • Figure 7i is a flow cytometry plot showing the expression of RORgT + on CD4 + T cells from colon tissues of W/DSS WT and W/DSS Nox4 -/- mice.
  • the bar graphs represent the percentage of CD4 + T cells (top right) and CD4 + RORgT + Th17 cells (bottom right) in the colons of WT and Nox4 -/- mice. Data are presented as mean ⁇ SD, and significance is *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.005, ****P ⁇ 0.001 compared to wild type (WT).
  • Figure 8 shows the related physiological characteristics of the colons of wild-type (WT) and Nox4 -/- mice.
  • Figure 8a shows the results of gene ontology (GO) analysis of the differentially expressed genes (DEGs) in the W/DSS WT group
  • Figure 8b shows the results of up-regulated gene sets among the differentially expressed genes (DEGs) in the W/DSS Nox4 - / - group.
  • Figure 8c shows the results of enzyme-linked immunosorbent assay for the active TGF- ⁇ levels in W/DSS WT or W/DSS Nox4 -/- colon tissue lysates. Data are expressed as mean ⁇ SD, and the significance is *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.005, ****P ⁇ 0.001 compared to the wild-type (WT).
  • Figure 9 shows that deletion of Nox4 impairs tissue repair following dextran sulfate sodium (DSS)-induced colitis.
  • Figure 9a is an experimental scheme for the recovery phase of DSS-induced murine colitis.
  • Figure 9d shows the results of hematoxylin and eosin staining of colonic tissues during the recovery phase after DSS-induced colitis in WT and Nox4 -/- mice
  • Figure 9e shows the results of quantification of colon length and disease activity index (DAI) scores.
  • DAI colon length and disease activity index
  • RNA-seq analysis To identify the underlying mechanisms contributing to the more severe inflammation and fibrosis observed in Nox4 -/- mice, we performed bulk RNA-seq analysis. A total of 5124 differentially expressed genes (DEGs) were compared across four groups [untreated (W/O) Nox4 -/- vs. W/O WT, treated (W/) Nox4 -/- vs. W/ WT, W/ WT vs. W/O WT, and W/ Nox4 -/- vs. W/O Nox4 -/- ]. Forty-two genes were co-upregulated and two genes were co-downregulated when comparing the treated and untreated groups.
  • DEGs differentially expressed genes
  • the 42 co-regulated genes including T cell-related genes (CD3e and Tnfrsf9), fibroblast-activating factors (Itg2a and Vegf), inflammasome sensor (Nlrp3), and TGF- ⁇ target genes (Ano5 and Mucl1), were expressed at higher levels in Nox4 -/- compared with those in WT mice.
  • Nox2-null mice (Nox2 -/- ) were established using CRISPR/Cas9 technology with exon 4 deleted (Fig. 1a).
  • Nox4-null mice (Nox4 -/- ) were also established to test the influence of immune cells expressing Nox family members on the fibrotic features of DSS colitis (Fig. 1b).
  • mice were administered 2.5% DSS (MP Biochemicals, Santa Ana, CA, USA) in drinking water for 14 days to induce fibrosing colitis. A fresh DSS solution was prepared every 2 days.
  • mice were administered 2% (w/v) DSS in drinking water for 5 days, followed by a 10-day recovery period with autoclaved distilled water. Control mice were provided only autoclaved distilled water.
  • DAI Disease Activity Index
  • mice were sacrificed and perfused via the left ventricle with cold phosphate-buffered saline (PBS).
  • the colon tissues obtained were fixed overnight at 4°C with cold 4% paraformaldehyde, processed into a “Swiss roll” shape, dehydrated, embedded in paraffin, and sectioned to 5 ⁇ m thickness.
  • the sections were deparaffinized three times with xylene for 20 min each, three times with absolute EtOH for 10 min each, twice with 90% EtOH for 10 min each, and 75% EtOH for 10 min, and then stained with hematoxylin and eosin.
  • the tissue slides were then dehydrated and mounted using Shandon synthetic mount solution (Thermo Scientific).
  • the sections were treated with 3% H 2 O 2 for 30 min to block endogenous peroxidase and then reacted with serum-free reagent (Dako) at room temperature for 2 h. Afterwards, the sections were treated with the primary antibodies in Table 1 overnight at 4°C, washed with PBS, and treated with horseradish peroxidase (HRP)-labeled secondary antibodies (Dako) at room temperature for 20 minutes. Liquid DAB+ staining was performed for target retrieval, and counterstained with Mayer's hematoxylin (Dako).
  • HRP horseradish peroxidase
  • TGF- ⁇ Expression levels of active TGF- ⁇ were measured using the TGFbR1 kinase ELISA system according to the manufacturer's instructions (Promega, Madison, WI, USA). To measure active TGFbR1, freshly isolated colon lysates were activated with 1 N HCl.
  • RNA sample RNA sample was hybridized with an oligo-dT primer containing an Illumina-compatible sequence at the 5'-end, and reverse transcription was performed. After the RNA template was isolated, second-strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at the 5'-end.
  • the double-stranded library was purified using magnetic beads to remove all reaction components, and the library was amplified to add the entire adapter sequence required for cluster generation.
  • the library was purified from PCR components, and high-throughput sequencing was performed with single-end 75 bp sequencing using the NextSeq 500 platform (Illumina, Inc., San Diego, CA, USA).
  • QuantSeq 3'mRNA-Seq reads were aligned using Bowtie2.
  • Bowtie2 indices were generated from representative transcriptome sequences for alignment to genome assembly sequences or genome and transcriptome. Alignment files were used to assemble transcriptomes and estimate abundance and detect DEGs between non-DSS-treated WT (W/O WT), non-DSS-treated Nox4 -/- (W/O Nox4 -/- ), DSS-treated WT (W/DSS WT), and DSS-treated Nox4 -/- (W/DSS Nox4 -/- ) distal colon samples.
  • Samples were defined as DEGs using a 2-fold cutoff value and P value based on the number of unique and multiple alignments considering the coverage of bedtools.
  • Read count data were processed based on the Quantile Normalization method using the EdgeR package in R software (R Development Core Team, 2016) with Bioconductor, and gene classification for functional investigation was based on searches of DAVID (/david.abcc.ncifcrf.gov/) and Medline databases (/www.ncbi.nlm.nih.gov/).
  • Data mining and graphical visualization were performed using ExDEGA (Ebiogen Inc., Korea).
  • RNAlaterTM stabilization solution Invitrogen, Thermo Fisher Scientific, Inc.
  • TRIzol reagent Invitrogen, Thermo Fisher Scientific
  • Contaminating genomic DNA was removed using Recombinant DNase1 (Takara Bio Inc., Shiga, Japan), and RNA was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific).
  • a total of 1 ⁇ g of template RNA was reverse transcribed using the ImProm-IITM reverse transcription system (Promega).
  • the primers used for qPCR are listed in Table 2 below.
  • the obtained colon tissues were treated with RIPA solution (Sigma Aldrich), and the purified proteins were loaded onto 8-15% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore). Protein expression was detected using the primary antibodies in Table 1 above, and after incubation with HRP-labeled secondary antibodies, the expression levels of specific proteins were measured using Signal West Femto Maximum Sensitivity Substrate (Thermo Scientific) together with a LAS chemiluminescence imaging system (Amersham).
  • Nox4 protects tissues from DSS-induced colitis.
  • Nox4 In the development of fibrosis in DSS-induced colitis, we confirmed Nox4 expression in mouse tissues. Immunohistochemistry was performed on normal colon and inflamed colon tissues treated with DSS. Nox4 was detected in the interstitial region in normal colon (Fig. 1c, upper left), whereas the levels of Nox4 transcript were significantly increased in the mucosal and submucosal regions in the colitis model administered with 2.5% DSS (Fig. 1c, lower left). Loss of Nox4 increased ROS production in both control (Fig. 1e) and DSS-induced colitis model (Fig. 1f) mice, suggesting that Nox4 elevation may be associated with disease progression.
  • Nox4 -/- mice After inducing the inflammatory phase of colitis by DSS treatment (W/DSS) (Fig. 2a), Nox4 -/- mice showed aggravated inflammation with rapid body weight loss of about 20-30% (Fig. 2b) and reduced survival rate (Fig. 2c), shorter colon length, and more severe inflammatory areas in the distal colon, including inflammatory cell infiltration and crypt loss (Figs. 2e, 2f) compared with wild-type mice (WT). W/DSS Nox4 -/- mice showed higher disease activity index (DAI) scores from day 1 to 14 than the W/DSS WT group (Fig. 2g).
  • DAI disease activity index
  • Sirius red staining showed increased intestinal fibrosis (Fig. 3c, top), consistent with increased expression of general fibrosis markers such as Col1a1 and Col3a1 (Fig. 3e) detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
  • RT-qPCR reverse transcription-quantitative polymerase chain reaction
  • mRNA levels of inflammatory cytokines tumor necrosis factor (Tnf) and interleukin-1beta (Il1 ⁇ ) were significantly increased in colitis-induced Nox4 -/- mice compared to colitis-induced WT mice at the same time points (Fig. 3d).
  • Nox4 -/- mice showed an increase in F4/80 + macrophages and Ki-67 + proliferating cells (Fig. 4a).
  • mRNA levels of fibrosis-associated markers and inflammatory cytokines Tnf and Il1 ⁇ were also increased in Nox4 -/- mice (Fig. 3d, Fig. 3e).
  • RNA-seq analysis To identify the underlying mechanisms contributing to the more severe inflammation and fibrosis observed in Nox4 -/- mice, we performed bulk RNA-seq analysis (Fig. 5a). A total of 5124 differentially expressed genes (DEGs) were compared across four groups [untreated (W/O) Nox4 -/- vs. W/O WT, treated (W/) Nox4 -/- vs. W/ WT, W/ WT vs. W/O WT, and W/ Nox4 -/- vs. W/O Nox4 -/- ]. Comparing the treated and untreated groups, 42 genes were co-upregulated and 2 genes were co-downregulated (Fig. 5b).
  • the 42 co-regulated genes including T cell-related genes (CD3e and Tnfrsf9), fibroblast-activating factors (Itg2a and Vegf), inflammasome sensor (Nlrp3), and TGF- ⁇ target genes (Ano5 and Mucl1), were expressed at higher levels in Nox4 -/- compared with those in WT mice.
  • Nox4 deficiency aggravates colitis by promoting intestinal inflammation through inducing the TGF- ⁇ pathway.
  • W/DSS Nox4 -/- mice showed significant expression levels of TGF- ⁇ -related genes, such as Asap3 and Tgfbr1, which are positive regulators of the TGF- ⁇ pathway; inflammatory-mediated Th17 cell differentiation genes, such as Plekha and Batf; Fibrosis-related genes such as Acta1 and Myo6, which promote collagen synthesis, were also increased.
  • TGF- ⁇ -related genes such as Asap3 and Tgfbr1, which are positive regulators of the TGF- ⁇ pathway
  • inflammatory-mediated Th17 cell differentiation genes such as Plekha and Batf
  • Fibrosis-related genes such as Acta1 and Myo6, which promote collagen synthesis
  • ulcerative colitis (UC)-related genes such as Chic1, Ido1, and Reg3g
  • immune infiltration-related genes such as Tmco3, Orai2, and Saa2 were upregulated in W/DSS WT mice (Fig. 7c).
  • W/DSS Nox4 -/- colons Compared to W/DSS WT colons (Fig. 7d, e).
  • GO analysis of W/DSS WT or W/DSS Nox4 -/- DEGs showed enrichment of upregulated W/DSS WT DEGs in tissue repair mechanisms such as proliferation, wound healing, and defense response in terms of cellular components associated with colitis-mediated functions such as innate immune response, biological stimuli, and cytokines.
  • up-regulated W/DSS WT DEGs were activated in the biological processes of endoplasmic reticulum stress and tissue repair mechanisms associated with intestinal inflammation, such as innate immune response and cytokine production.
  • DEGs were enriched in molecular function GO terms included in inflammatory bowel disease (IBD) pathophysiology-related gene sets, such as calcium ion binding, peptidase activity and signal transduction-related functions (Fig. 8a).
  • IBD inflammatory bowel disease
  • W/DSS Nox4 -/- DEGs showed different functional patterns.
  • tissue damage gene sets such as cell death, inflammation and injury.
  • the most enriched gene set was identified as tissue fibrosis-related gene set, such as catalytic complex, cytoskeletal part and ribonucleoprotein complex.
  • DEGs In terms of molecular function terms, highly enriched DEGs were identified as UC-related oncogene sets, such as protein binding, protein kinase activity, histone acetyltransferase activity and oxidoreductase activity (Fig. 8b).
  • DEGs of W/DSS WT were enriched in the Toll-like receptor pathway, JAK-STAT pathway, and mitogen-activated protein kinase pathway
  • DEGs of W/DSS Nox4 -/- were enriched in gene sets of the TGF- ⁇ pathway, and T cell receptor and rapamycin pathways (Fig. 7f).
  • the heatmap confirmed that the expression level of TGF- ⁇ target genes in DEGs of W/DSS Nox4 -/- group was significantly increased compared to that of DEGs of W/DSS group (Fig. 7g).
  • TGF- ⁇ signaling and its associated fibrosis and T cell signaling are persistently increased in the colons of Nox4-deficient mice, contributing to the maintenance of more severe inflammation.
  • Western blotting showed that TGF- ⁇ and TGFbR1 protein levels were elevated in W/DSS Nox4 -/- colon lysates compared to W/DSS WT colon lysates (Fig. 7h).
  • ELISA analysis also confirmed that active TGF- ⁇ levels were 2-fold increased in W/DSS Nox4 -/- colons compared to W/DSS WT colons (Fig. 8c).
  • the researchers have elucidated that genetic deletion of Nox4 leads to the progression of more severe fibrosing colitis and subsequent failure of recovery.
  • Loss of Nox4 increased oxidative stress in colonic tissue and subsequently permanently activated canonical TGF- ⁇ signaling.
  • it affected the differentiation and proliferation of T cell lineages (Treg and Th17 cells) mediated by TGF- ⁇ , and the progression of fibrosis in colitis. Therefore, the present invention is expected to be greatly utilized in predicting whether fibrosis progresses in inflammatory bowel disease and suppressing it.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne la découverte d'une cible pour inhiber la fibrose dans une maladie intestinale inflammatoire, et la prédiction de la progression de la fibrose dans une maladie intestinale inflammatoire à l'aide de celle-ci. Les présents inventeurs, qui sont des chercheurs, ont découvert que les transcrits NOX4 sont détectés à de faibles niveaux dans un côlon normal, mais que ces transcrits sont surexprimés chez les patients atteints de fibrosténose pharmacorésistante due à la rectocolite hémorragique ou à la maladie de Crohn (MC) et dans un modèle de colite induite par le sulfate de dextran sodique (DSS) chez la souris. Ils ont donc émis l'hypothèse que NOX4 contribue à la fibrose intestinale causée par la colite ulcéreuse et que la signalisation TGF-β est nécessaire pour induire le développement de la fibrose. Afin de vérifier cette hypothèse, les présents inventeurs ont développé des souris déficientes en Nox4 (Nox4-/-) avec une colite induite par le DSS, et ont étudié l'effet de Nox4 sur la réponse à la lésion intestinale. Cette étude devrait être largement utilisée pour prédire la progression de la fibrose dans les maladies intestinales inflammatoires et l'inhiber.
PCT/KR2024/005979 2023-05-03 2024-05-03 Découverte d'une cible pour inhiber la fibrose dans une maladie intestinale inflammatoire Ceased WO2024228570A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2023-0057549 2023-05-03
KR20230057549 2023-05-03

Publications (1)

Publication Number Publication Date
WO2024228570A1 true WO2024228570A1 (fr) 2024-11-07

Family

ID=93333135

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2024/005979 Ceased WO2024228570A1 (fr) 2023-05-03 2024-05-03 Découverte d'une cible pour inhiber la fibrose dans une maladie intestinale inflammatoire

Country Status (2)

Country Link
KR (1) KR20240161931A (fr)
WO (1) WO2024228570A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120141461A1 (en) * 2009-07-17 2012-06-07 The Regents Of The University Of Michigan Compositions and methods for diagnosing and treating fibrotic disorders
WO2019018571A1 (fr) * 2017-07-18 2019-01-24 Washington University Procédés et utilisations de biomarqueurs de maladie intestinale inflammatoire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120141461A1 (en) * 2009-07-17 2012-06-07 The Regents Of The University Of Michigan Compositions and methods for diagnosing and treating fibrotic disorders
WO2019018571A1 (fr) * 2017-07-18 2019-01-24 Washington University Procédés et utilisations de biomarqueurs de maladie intestinale inflammatoire

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAN CHENYANG, SHENG YONGJIA, WANG JIN, ZHOU XIAOHONG, LI WENYAN, ZHANG CAIQUN, GUO LI, YANG YI: "NOX4 promotes mucosal barrier injury in inflammatory bowel disease by mediating macrophages M1 polarization through ROS", INTERNATIONAL IMMUNOPHARMACOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 104, 1 March 2022 (2022-03-01), NL , pages 108361, XP093230452, ISSN: 1567-5769, DOI: 10.1016/j.intimp.2021.108361 *
JARMAN ELIZABETH R., KHAMBATA VALERIE S., COPE CLAIRE, JONES PETER, ROGER JAN, YE LI YUN, DUGGAN NICHOLAS, HEAD DENISE, PEARCE AND: "An Inhibitor of NADPH Oxidase-4 Attenuates Established Pulmonary Fibrosis in a Rodent Disease Model", AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY., AMERICAN LUNG ASSOCIATION, NEW YORK, NY, US, vol. 50, no. 1, 1 January 2014 (2014-01-01), NEW YORK, NY, US , pages 158 - 169, XP093230456, ISSN: 1044-1549, DOI: 10.1165/rcmb.2013-0174OC *
LEE YURA, KIM SUNG-HEE, JEONG HAENGDUENG, KIM KWANG H., JEON DONGHUN, CHO YEJIN, LEE DAEKEE, NAM KI TAEK: "Role of Nox4 in Mitigating Inflammation and Fibrosis in Dextran Sulfate Sodium–Induced Colitis", CMGH CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY, ELSEVIER, vol. 16, no. 3, 1 January 2023 (2023-01-01), pages 411 - 429, XP093230460, ISSN: 2352-345X, DOI: 10.1016/j.jcmgh.2023.05.002 *
STENKE EMILY, AVIELLO GABRIELLA, SINGH ASHISH, MARTIN SEAN, WINTER DES, SWEENEY BRIAN, MCDERMOTT MICHAEL, BOURKE BILLY, HUSSEY SEA: "NADPH oxidase 4 is protective and not fibrogenic in intestinal inflammation", REDOX BIOLOGY, ELSEVIER, NL, vol. 37, 1 October 2020 (2020-10-01), NL , pages 101752, XP093230447, ISSN: 2213-2317, DOI: 10.1016/j.redox.2020.101752 *

Also Published As

Publication number Publication date
KR20240161931A (ko) 2024-11-13

Similar Documents

Publication Publication Date Title
CN109576362B (zh) 阿尔茨海默诊治用标志物fam170a
KR20190095074A (ko) 뇌 종양 동물 모델 및 이의 제조 방법
WO2015093557A1 (fr) Nouveau gène de fusion en tant que gène responsable du cancer de l'estomac
Lehman et al. Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes
CN114457163B (zh) 一种间充质亚型胶质母细胞瘤的标志物及其应用
WO2017026843A1 (fr) Procédé de fourniture d'informations sur la leucémie myéloïde chronique
WO2020017914A1 (fr) Composition pour réguler la division ou la différenciation de cellules cancéreuses, contenant setdb1 ou un inhibiteur de celui-ci
CN114502189A (zh) 以屏障功能障碍为特征的疾病中的黏蛋白同种型
Qian et al. Involvement of USP7 in aggravating Kawasaki disease by promoting TGFβ2 signaling mediated endothelial-mesenchymal transition and coronary artery remodeling
Lopez et al. Gene expression profiling in lungs of chronic asthmatic mice treated with galectin‐3: downregulation of inflammatory and regulatory genes
US20230340623A1 (en) Mucins and isoforms thereof in diseases characterized by barrier dysfunction
WO2024228570A1 (fr) Découverte d'une cible pour inhiber la fibrose dans une maladie intestinale inflammatoire
KR102840605B1 (ko) SARS-CoV-2-유도성 패혈증의 진단 또는 예후 예측용 바이오마커
US20190316135A1 (en) Novel therapy
WO2019066263A1 (fr) Composition pharmaceutique permettant de prévenir ou de traiter une maladie dégénérative neuronale comprenant une protéine nckap1 ou un gène codant cette dernière
US11078535B2 (en) Presymptomatic micro RNA targets for treatment of neurodegeneration pathology
WO2022137964A1 (fr) Composition pharmaceutique pour la prévention ou le traitement de maladies du cartilage/de l'os/des articulations, et procédé de criblage de médicament pour la prévention ou le traitement de maladies du cartilage/de l'os/des articulations
Zhou et al. MRGPRX2-Mediated Mast Cell Activation Promotes Malignant Progression of Cutaneous Squamous Cell Carcinoma via IL-17A Release
WO2024195741A1 (fr) Agent thérapeutique et procédé d'examen pour hypertrophie cardiaque
EP4029516A1 (fr) Mucines et leurs isoformes utilisées dans des maladies caractérisées par un dysfonctionnement de la barrière
WO2022114878A1 (fr) Composition pour le diagnostic ou le traitement de la fibrose
Beilina et al. Inflammatory signaling differentially changes chromatin accessibility and gene expression of the PD-associated kinase LRRK2 between human and mice
WO2025127647A1 (fr) Composition pour la prévention ou le traitement d'une maladie infectieuse à coronavirus
WO2025084847A1 (fr) Biomarqueur pour diagnostiquer un état immunitaire et son utilisation
Chang et al. Parvimonas micra-derived SirTM: An ADP-ribosyltransferase as a novel driver in colorectal cancer progression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24800251

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE