Application of E3 ubiquitin ligase RNF219 in preventing or treating inflammatory diseases

Abstract

The invention relates to the technical field of biological medicines, in particular to application of E3 ubiquitin ligase RNF219 in preventing or treating inflammatory diseases. The invention provides an application of a substance for inhibiting the activity of RNF219 protein or a substance for reducing the content of RNF219 protein in preparation of drugs for preventing or treating inflammatory diseases, especially drugs for preventing or treating sepsis and colitis. According to the application, a mouse acute infection model is constructed to prove that RNF219 defect inhibits the inflammatory reaction degree and inflammatory injury induced by LPS (Lipopolysaccharide); in a mouse with DSS-induced colitis, the RNF219 defect can delay the disease progress of the mouse colitis and improve the inflammatory phenotype of the mouse. Therefore, the function of the RNF219 in natural immune response is defined, and the RNF219 has important scientific significance on prevention and treatment of immune inflammation related diseases such as sepsis and inflammatory bowel diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to the application of E3 ubiquitin ligase RNF219 in preventing or treating inflammatory diseases. BACKGROUND

[0002] Infection and inflammation are closely related to the pathological process of many diseases. How to prevent and treat infectious and inflammatory diseases is an important scientific problem that clinical and basic research pay attention to. In order to inhibit infection, clinicians usually use drugs to inhibit the proliferation of pathogens, so as to remove the cause. However, during the process of pathogen infection, the components of the pathogen and the toxins released by it spread from the primary site of the lesion to the whole body through the circulatory system, causing the immune system of the body to be in an overactive state, causing excessive immune inflammatory response, and further aggravating the damage of the body tissues and organs, and in severe cases, shock or multiple organ dysfunction will occur.

[0003] During the process of pathogen infection, the body's natural immune system can recognize "self" and "non-self" components through pattern recognition receptors (PRRs), and initiate downstream immune inflammatory response (Medzhitov R. et al., Nature. 2007; 449: 819-26). TLR (Toll like receptor) is one of the PRRs that people have recognized the earliest and also the most widely studied, which plays an important role in resisting pathogen invasion. For example, TLR4 located on the cell membrane can recognize bacterial lipopolysaccharide (LPS) and viral membrane glycoprotein, TLR5 recognizes bacterial flagellin, TLR1, 2 and 6 recognize bacterial lipoprotein; and TLR found in endosomes can detect pathogen-related nucleic acid substances, such as TLR3 can recognize viral double-stranded RNA, TLR7 and 8 recognize single-stranded RNA, and TLR9 recognizes ssDNA containing non-methylated CpG (Fitzgerald KA. et al., Cell. 2020; 180: 1044-1066).

[0004] Sepsis is a life-threatening clinical syndrome caused by the host's dysregulated response to infection, mainly caused by bacterial, fungal, viral and parasitic infections. The incidence of sepsis is about 0.3%, but its mortality rate is as high as 30% to 40%, and the mortality rate of septic shock is about 50%. The pathogenesis of sepsis is extremely complex, and studies have shown that the occurrence of sepsis is closely related to the activation of the TLR signaling pathway. Under the action of pathogen-derived pathogen-associated molecular patterns (PAMP), the TLR pathway is over-activated, inducing the release of a large amount of cytokines, further activating the immune system of the body, thereby triggering a 'cascade' cascade reaction, triggering a 'cytokine storm', leading to uncontrolled systemic inflammatory response. This pathophysiological process will cause serious damage to the body. A large number of inflammatory factors, on the one hand, damage the vascular endothelial cells, leading to increased vascular permeability, fluid and protein extravasation, causing tissue edema; on the other hand, it also activates the coagulation system, leading to microthrombosis, affecting tissue blood perfusion, and further causing multiple organ dysfunction syndrome (MODS), and severe cases will appear septic shock (van Deventer SJ. et al., Intensive Care Med. 2000; 26:S98-S102). Given the central role of immune inflammatory response in the development of sepsis, the use of immunomodulatory agents to treat sepsis has made great progress in clinical practice, but it still faces many challenges, and further focusing on the regulation mechanism of immune inflammatory response will bring hope for the prevention and treatment of sepsis.

[0005] Colitis is an intestinal disease characterized by inflammation, damage, and repair dysfunction of the intestinal mucosa. Inflammatory bowel disease is the most typical subtype of colitis, mainly including Crohn's disease and ulcerative colitis. The main clinical manifestations of inflammatory bowel disease are abdominal pain, diarrhea, mucous and bloody stool, and persistent inflammation may increase the risk of intestinal fibrosis and cancer (Gros B. et al., JAMA. 2023; 330: 951-965). The occurrence of inflammatory bowel disease is closely related to abnormal intestinal immune function. The core of its pathophysiology is the imbalance of intestinal immune homeostasis. On the one hand, immune cells in the intestine over-secrete pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin 6 (IL-6), and IL-17 under the action of genetic and environmental factors, on the other hand, the function of anti-inflammatory immune cells such as regulatory T cells (Treg) is impaired, thereby triggering persistent excessive inflammatory response in the intestine, ultimately leading to chronic damage and dysfunction of the intestinal mucosa (Johansson ME. et al., Nat Rev Gastroenterol Hepatol. 2013; 10: 352-361). In addition, a large number of studies have shown that abnormal activation of the TLR signaling pathway is closely related to the occurrence and development of colitis. For example, LPS released by intestinal symbiotic bacteria under the condition of intestinal immune imbalance will lead to the activation of the TLR4 signaling pathway, thereby causing the production of a large number of pro-inflammatory cytokines; CpG DNA produced by intestinal bacteria or host cell mitochondrial DNA may cause abnormal activation of the TLR9 signaling pathway, promote the secretion of type I interferon and TNF, and lead to the occurrence of intestinal inflammation (Ai L. et al., Gut. 2021; 70: 2261-2272; Obermeier F. et al., Gastroenterology. 2005; 129: 913-927). Currently, there are cases of using anti-TNF monoclonal antibodies, anti-IL-17 antibodies, and immunomodulators (such as azathioprine, which regulates T cell function) to treat inflammatory bowel disease in clinical practice, further indicating that further analysis of the mechanism of immune inflammatory response in the pathogenesis of colitis is expected to provide new targets for the diagnosis and treatment of colitis.

[0006] The ubiquitin-proteasome system (UPS) is one of the important ways of post-translational modification of cells, and is also the main pathway for cells to exert protein degradation function (Li J. et al., Cell Mol Immunol. 2016; 13: 560-576). In this system, E1 ubiquitin activating enzyme, E2 ubiquitin binding enzyme and E3 ubiquitin ligase work together to selectively degrade intracellular target proteins, affecting processes such as cell transcription, translation, endocytosis, receptor activation and subcellular localization. Among them, E3 ubiquitin ligase can recruit E2 and specific protein substrates, and connect single ubiquitin molecules, polyubiquitin molecules, ubiquitin chains, etc. to the specific site of the substrate protein, thus playing a key role in the ubiquitination modification process.

[0007] TLR activates NF-κB (Nuclear factor κB) through downstream signal transduction after activation, and abnormal activation of NF-κB is closely related to various infections and inflammatory diseases (Apetoh L. et al., Nat Med. 2007; 13: 1050-9). At present, the ubiquitination regulation of NF-κB transcription factor mostly focuses on the ubiquitin degradation pathway of NF-κB transcription factor, thus playing a negative regulatory function in the NF-κB signaling pathway. For example, with the help of COMMD1 (Copper metabolism MURR1 domain-containing 1), the ubiquitin ligase complex ECS (Elongin-B-elongin-C-cullin-SOCS1) promotes the ubiquitination degradation of P65. Nuclear protein PDLIM2 (PDZ and LIM domain protein 2) can promote P65 to form PML (Promyelocytic leukemia protein) nuclear bodies in the nucleus, inhibit the binding of P65 and DNA, and promote the degradation of P65 in the nucleus. RNF182 can promote the K48-linked polyubiquitination of P65, thereby inhibiting the inflammatory response triggered by TLR3 (Nakagawa R. et al., Immunity. 2002; 17: 677-87; Geng H. et al., EMBO Rep. 2009; 10: 381-6; Tanaka T. et al., Nat Immunol. 2007; 8: 584-91; Cao Y. et al., FEBS Lett. 2019; 593: 3210-3219). However, as of now, there are few reports on E3 ubiquitin ligase regulating the activation of NF-κB transcription factor through ubiquitin modification.

[0008] RNF219 is a typical E3 ubiquitin ligase molecule containing a RING domain. Previous studies have shown that RNF219 plays an important regulatory function in life activities including DNA replication, cell differentiation and development, and tumor occurrence and development, etc. by virtue of its ubiquitin modification activity. RNF219 can inhibit mRNA degradation by inhibiting CCR4 (Carbon catabolite repression 4)-NOT (Negative on TATA-less) complex-mediated de-adenylation. In addition, in a liver cancer bone metastasis model, researchers found that RNF219 can promote YAP1 / β-Catenin-mediated LGALS3 (Lectin galactoside-binding soluble 3) promoter epigenetic modification by regulating α-Catenin degradation, thereby promoting tumor metastasis. In the innate immune response, Hwang et al. found that RNF219 inhibits the ubiquitination degradation of SIRT1 (sirtuin-1) by binding to deacetylase SIRT1, thereby inhibiting the inflammatory response induced by lipopolysaccharide LPS (Poetz F. et al., Nat Commun. 2021; 12: 7175; Zhang S. et al., Adv Sci (Weinh). 2020; 8: 2001961; Hwang JS. et al., Br J Pharmacol. 2020; 177: 4601-4614).

[0009] At present, the research on the function of RNF219, especially its research in the innate immune response, is still in its infancy. Exploring the regulatory role and molecular mechanism of RNF219 in the innate immune response triggered by classical pattern recognition receptors and inflammatory response has important scientific significance for the prevention and treatment of immune inflammatory related diseases such as sepsis and inflammatory bowel disease. SUMMARY

[0010] Therefore, the present application provides the application of E3 ubiquitin ligase RNF219 in preventing or treating inflammatory diseases.

[0011] In a first aspect, the present application provides the application of a substance inhibiting the activity of RNF219 protein or a substance reducing the content of RNF219 protein in the preparation of a drug for preventing or treating inflammatory diseases.

[0012] In some embodiments, the inflammatory disease includes sepsis and colitis, preferably the colitis is inflammatory bowel disease.

[0013] In some embodiments, the substance that inhibits the activity of RNF219 protein is a protein, polypeptide, or small molecule compound that inhibits the function of RNF219 protein; or, the substance that reduces the content of RNF219 protein is a substance that inhibits the synthesis of RNF219 protein, promotes the degradation of RNF219 protein, or knocks down or eliminates the RNF219 gene.

[0014] In some embodiments, the substance that inhibits the activity of the RNF219 protein includes an RNF219 antibody.

[0015] In some embodiments, the substance that inhibits RNF219 protein synthesis includes a substance that inhibits the expression of the gene encoding the RNF219 protein, preferably a substance that silences the gene encoding the RNF219 protein.

[0016] In this application, the substance that silences the gene encoding the RNF219 protein can be a substance such as miRNA, siRNA, dsRNA, shRNA, etc.

[0017] In some embodiments, the substance that knocks down or eliminates the RNF219 gene includes an RNF219 gene knockout system.

[0018] In this application, knockout means that the host cell carrying the knockout substance does not produce the functional protein product of the gene. The knockout substance can be the removal of all or part of the coding gene sequence, the introduction of a frameshift mutation to prevent the production of functional proteins, the removal or alteration of regulatory components (e.g., promoter editing) to prevent the coding gene sequence from being transcribed, or the prevention of translation by binding to mRNA, etc. Typically, knockout is performed at the genomic DNA level, so that the cell's offspring also permanently carry the knockout.

[0019] Secondly, this application provides a product for the prevention or treatment of inflammatory diseases, wherein the active ingredient of the product is a substance that inhibits the activity of RNF219 protein or a substance that reduces the content of RNF219 protein.

[0020] In some embodiments, the substance that inhibits the activity of RNF219 protein is a protein, polypeptide, or small molecule compound that inhibits the function of RNF219 protein; or, the substance that reduces the content of RNF219 protein is a substance that inhibits the synthesis of RNF219 protein, promotes the degradation of RNF219 protein, or knocks down or eliminates the RNF219 gene.

[0021] Thirdly, this application also provides the application of RNF219 as a target in screening drugs for the prevention or treatment of inflammatory diseases. The application is that if a substance has the function of inhibiting RNF219 protein synthesis or promoting RNF219 protein degradation or knocking down or knocking out the RNF219 gene, then the substance is a candidate drug for the prevention or treatment of inflammatory diseases.

[0022] In some embodiments, the inflammatory disease comprises sepsis and colitis, preferably the colitis is inflammatory bowel disease.

[0023] Compared with the prior art, the present application can inhibit the production of LPS-induced macrophage pro-inflammatory related cytokines and type I interferons by targeting knockdown or knockout of RNF219, and the RNF219 small interfering RNA can inhibit the inflammatory response of macrophages to LPS; at the same time, after targeting knockdown or knockout of RNF219, the activation of NF-κB signaling pathway of macrophages under LPS stimulation is significantly inhibited, and the nuclear amount of P65 is significantly reduced, while the activation of MAPK, TBK1-IRF3 signaling pathway is not significantly changed. In addition, it is proved that RNF219 deficiency can inhibit the degree of LPS-induced inflammatory response and inflammatory damage by constructing a mouse acute infection model; in DSS-induced colitis mice, RNF219 deficiency can delay the disease progression of mouse colitis and improve the inflammatory phenotype of mice. Therefore, the present application clearly defines the function of RNF219 in the innate immune response, which has important scientific significance for the prevention and treatment of immune inflammation-related diseases such as sepsis and inflammatory bowel disease. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present application, the drawings required in the description of the specific embodiments will be briefly introduced as follows.

[0025] Figure 1 The detection results of the ability of macrophages to produce inflammatory cytokines and type I interferons after targeting knockdown or knockout of RNF219. Among them, Figure 1 A and B in the table are the expression of RNF219 in LPS-stimulated mouse macrophages detected by fluorescence quantitative PCR and Western blotting technology; Figure 1 C in the table is the expression level of inflammatory cytokines and type I interferons in LPS-stimulated mouse macrophages after targeting knockdown of RNF219 by small interfering RNA detected by fluorescence quantitative PCR; Figure 1 D in the table is the expression level of inflammatory cytokines and type I interferons in LPS-stimulated macrophages of RNF219-deficient mice detected by fluorescence quantitative PCR; Figure 1 E in the table is the secretion of inflammatory cytokines and type I interferons by RNF219-deficient mouse macrophages detected by ELISA. * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001, n.s. is no statistical difference, PM is peritoneal macrophage, BMDM is bone marrow-derived macrophage, Ctrl is control group, Rnf219 + / +RNF219 wild type control group, Rnf219 - / - RNF219 expression deficiency group.

[0026] Figure 2 The degree of activation of the NF-κB signaling pathway in macrophages after targeted knockdown or knockout of RNF219 was detected. Among them, Figure 2 In the middle A, after the small interfering RNA targeted knockdown of RNF219, the mouse macrophages were stimulated using LPS, and the expression and phosphorylation of key molecules in the NF-κB, TBK1-IRF3 and MAPK signaling pathways were detected using Western blotting technology; Figure 2 In the middle B and C, the macrophages of wild type and RNF219 deficient mice were stimulated using LPS, and the expression and phosphorylation of key molecules in the NF-κB, TBK1-IRF3 and MAPK signaling pathways were detected using Western blotting technology, and the nuclear entry of P65 was detected. Figure 2 In the middle D, the wild type and RNF219 deficient RAW264.7 knockout cells were stimulated using LPS, and the nuclear entry of P65 was observed using fluorescence microscopy. NC is the control group, Rnf219 + / + RNF219 wild type control group, Rnf219 - / - RNF219 expression deficiency group.

[0027] Figure 3 RNF219 can promote the activation of NF-κB and Ifnb luciferase reporter gene detection results. Among them, Figure 3 In the middle A, the HEK293T cells were transiently transfected with Renilla luciferase internal reference gene, PRR signaling pathway key linker molecules (including MyD88, TRIF, cGAS, STING, MAVS, RIG-I, IRF3, etc.), RNF219 and NF-κB, Ifnb or AP-1 luciferase reporter gene expression vector, and fluorescence intensity detection was performed. Figure 3 In the middle B, RNF219 was gradient transfected in HEK293T cells, and the rest was the same as Figure 3 A, and fluorescence intensity detection was performed. * represents P<0.05, *** represents P<0.001, **** represents P<0.0001, Mock is the blank vector group, and RNF219 is the RNF219 expression vector group.

[0028] Figure 4 RNF219 deficient mice have weaker LPS-induced immune inflammatory response. Among them, Figure 4 In the middle A, H&E staining analysis of mouse lung after LPS intraperitoneal injection; Figure 4 In the middle B, H&E staining analysis of mouse kidney after LPS intraperitoneal injection; Figure 4The levels of inflammatory cytokines and type I interferon in the serum of wild-type and RNF219-deficient mice were detected by ELISA after intraperitoneal injection of LPS. Figure 4 The survival of wild-type and RNF219-deficient mice after intraperitoneal injection of LPS was analyzed using Kaplan-Meier survival analysis. * represents P<0.05, *** represents P<0.001, ns indicates no statistical difference, Ctrl represents the control group, and Rnf219... + / + This is the wild-type control group for RNF219. - / - This group is characterized by RNF219 expression deficiency.

[0029] Figure 5 RNF219-deficient mice showed a lower response to DSS-induced acute colitis. Figure 5 In Figure A, the daily weight (compared to the initial weight, set to 100%), diarrhea score, and bloody stool score of mice during the induction of colitis using 3% DSS are recorded. Figure 5 Image B shows gross morphological photographs of the colon of control (fed with normal drinking water) and colitis (induced by 3% DSS for 9 days); Figure 5 In the middle section (C), the colon length of control (fed with normal drinking water) and colitis (induced by 3% DSS for 9 days) mice was statistically analyzed. Figure 5 In the middle section (D), H&E staining analysis was performed on colonic tissue sections from mice with colitis (induced by 3% DSS for 9 days). Figure 5 E represents the levels of inflammatory cytokines and type I interferon in the serum of mice with colitis (induced by 3% DSS for 9 days) detected by ELISA. * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001, ns represents no statistical difference, Naive represents the control group fed with normal drinking water, and Rnf219 + / + This is the wild-type control group for RNF219. - / - This group is characterized by RNF219 expression deficiency. Detailed Implementation

[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 application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer. Unless otherwise specified, the test reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods unless otherwise specified.

[0031] Example 1 Primary macrophages derived from mouse peritoneum were obtained by intraperitoneal injection of 3% sodium thioglycolate broth into mice. Four days after injection, the mice were sacrificed, and serum-free DMEM medium was injected into the peritoneum. After repeated flushing, the peritoneal lavage fluid was centrifuged, and the precipitate contained the desired primary macrophages. Primary macrophages derived from mouse bone marrow were obtained by flushing out femoral and tibial bone marrow from mice into serum-free DMEM medium, lysing the erythrocytes with Tris-NH4Cl, and then inducing the differentiation of monocytes in the bone marrow into mature macrophages using M-CSF (macrophage colony-stimulating factor, 20 ng / mL). Six days later, the induced macrophages were digested and plated for culture.

[0032] Primary macrophages derived from mouse peritoneum and bone marrow were stimulated with LPS (purchased from Merck). Total RNA was then collected and reverse transcribed to detect RNF219 gene expression. Using interfering RNA (RNAi) technology, RNF219 expression was interfered with in wild-type mouse peritoneum and bone marrow macrophages. Cells were then stimulated with LPS (100 ng / mL) at 1 h, 2 h, 4 h, 8 h, and 12 h. Cells treated with Control siRNA (Ctrl siRNA) served as controls. The mRNA expression levels of Ifnb, Tnf, Il6, and Il10 were detected using quantitative real-time PCR. Primers used are shown in Table 1. Western blotting was used to detect the expression of related proteins in the cells. Type I interferon and pro-inflammatory cytokines in the supernatant were detected using an ELISA kit.

[0033] Table 1 Primer sequences for real-time PCR

[0034] The results showed that both the mRNA and protein of RNF219 were downregulated after LPS stimulation. Figure 1 (A and B). It was found that after interfering with RNF219 expression, the expression of Ifnb, Tnf, and Il6 significantly decreased, while the expression of Il10 was upregulated (…). Figure 1 (C). Subsequently, primary macrophages derived from wild-type and RNF219 myeloid knockout mice were treated under the same conditions. It was found that after RNF219 targeted knockout, the expression of Ifnb, Tnf, and Il6 was downregulated, while the expression of Il10 was upregulated. Figure 1 (D). The levels of IFNβ, TNF, and IL-6 in the cell supernatant after stimulation were detected using ELISA. It was found that the levels of TNF and IL-6 in the supernatant of RNF219 knockout cells were significantly lower than those in the control group. Figure 1 E).

[0035] Therefore, the ability of macrophages to produce inflammatory cytokines and type I interferon is reduced after targeted knockdown or knockout of RNF219.

[0036] Example 2 Using siRNA technology, the expression of RNF219 was interfered in macrophages derived from the peritoneal cavity of wild-type mice. The small interfering RNA sequences used are shown in Table 2.

[0037] Table 2 Small interfering RNA sequences

[0038] Cells were then stimulated with LPS (100 ng / mL) at 15 min and 30 min, with NC-treated cells used as controls. Western blotting was used to detect the expression and phosphorylation of key molecules in the NF-κB, TBK1-IRF3, and MAPK signaling pathways. The results showed that activation of the NF-κB signaling pathway was significantly inhibited. Figure 2 (A). Subsequently, primary macrophages derived from wild-type and RNF219 myeloid knockout mice were treated under the same conditions. Western blotting was used to detect the expression and phosphorylation of key molecules in the NF-κB, TBK1-IRF3, and MAPK signaling pathways, and p65 nuclear translocation was also examined. It was found that activation of the NF-κB signaling pathway was significantly inhibited, and p65 nuclear translocation was significantly reduced (…). Figure 2 (B and C). Wild-type and RNF219-deficient RAW264.7 knockout cells were stimulated with LPS, and P65 nuclear translocation was observed using fluorescence microscopy. It was found that P65 nuclear translocation was significantly inhibited after RNF219 knockout. Figure 2 D).

[0039] Therefore, after targeted knockdown or knockout of RNF219, the activation of the NF-κB signaling pathway in macrophages under LPS stimulation was significantly inhibited, and the amount of P65 entering the nucleus was significantly reduced, while the activation of the MAPK and TBK1-IRF3 signaling pathways was not significantly changed.

[0040] Example 3 HEK293T cells were transiently transfected with the Renal luciferase internal control gene, key PRR signaling pathway adaptor molecules (including MyD88, TRIF, cGAS, STING, MAVS, RIG-I, IRF3, etc.), RNF219, and NF-κB, Ifnb, or AP-1 luciferase reporter gene expression vectors. Forty-eight hours after transfection, cells were lysed using 1×PLB (passive lysis buffer), and fluorescence detection reagents were added to measure the luminescence value of the experimental reporter genes. The reaction was then terminated with stop solution, and the luminescence value of the internal control was measured.

[0041] RNF219 can promote MyD88-induced NF-κB reporter gene activation ( Figure 3 A). For the Ifnb reporter gene, RNF219 can promote MAVS, IRF3, and MyD88-induced Ifnb reporter gene activation, but has no significant effect on the AP-1 reporter gene. Figure 3 A). Furthermore, with increasing RNF219 transfection levels, the activation level of the MyD88-induced NF-κB reporter gene gradually increased, and the activation level of the MAVS and IRF3-induced Ifnb reporter genes also increased to some extent with increasing RNF219 transfection levels. Figure 3 B), while gradient transfection of RNF219 still had no significant effect on the AP-1 reporter gene (B). Figure 3 B) suggests that RNF219 can promote the activation of the NF-κB signaling pathway downstream of MyD88 and has a positive regulatory effect on the transcription of the Ifnb gene involved by MAVS and IRF3.

[0042] These results indicate that RNF219 can promote the activation of NF-κB and Ifnb luciferase reporter genes.

[0043] Example 4 An acute infection model was established in wild-type and RNF219 myeloid knockout mice by intraperitoneal injection of LPS (10 mg / kg), with intraperitoneal injection of PBS as a control. Twelve hours after injection, mice were euthanized by cervical dislocation, and lungs, kidneys, and other organs were dissected and fixed with paraformaldehyde. Histopathological analysis of the tissues and organs was performed using H&E staining and CD45 assay. + F4 / 80 + Ly6G +The infiltration of immune inflammatory cells was analyzed using various detection indicators. Ocular venous blood was collected from mice, and serum was separated. The levels of inflammatory cytokines and type I interferon in the serum were detected using an ELISA kit. Wild-type and RNF219 myeloid knockout mice were intraperitoneally injected with LPS (10 mg / kg), and the survival status of the mice was statistically analyzed.

[0044] H&E histopathological staining revealed that, compared with wild-type mice, RNF219 myeloid knockout mice showed no significant differences in lung and kidney lesions after intraperitoneal injection of PBS compared with the control group. However, after intraperitoneal injection of LPS, the degree of inflammatory cell infiltration in the lungs and kidneys was lower, and there was less inflammatory exudate in the lung interstitium. Figure 4 (A and B). Furthermore, ELISA was used to detect the levels of IFNβ, TNF, and IL-6 in serum, revealing that the levels of inflammatory cytokines and type I interferon in the serum of RNF219-deficient mice injected intraperitoneally with LPS were significantly lower than those in control mice. Figure 4 (C). Kaplan-Meier survival analysis results indicated that the survival rate of RNF219 myeloid-deficient mice after intraperitoneal injection of LPS was significantly improved compared with wild-type mice. Figure 4 (D).

[0045] These results demonstrate that RNF219 deficiency inhibits the extent of LPS-induced inflammatory response and inflammatory damage.

[0046] Example 5 Sodium dextran sulfate (DSS, purchased from MP Biomedicals) was diluted to 3% with pure water and provided to mice for free drinking, with the solution changed every 3 days. Control mice were fed normal drinking water. During the induction of colitis, mouse weight, stool characteristics, and blood in stool were recorded daily. Normal stool characteristics were scored as 0 points, soft stool as 1 point, mucous stool as 3 points, and watery stool as 4 points. Normal stool color was scored as 0 points, light blue occult blood indicator as 1 point, blue occult blood indicator as 2 points, dark blue occult blood indicator as 3 points, and visible blood in stool as 4 points. On day 9 of colitis induction, mice were euthanized by cervical dislocation, and the colon was dissected and separated. The gross morphology of the colon was photographed, and the colon length was measured. The distal colon tissue was separated and fixed with paraformaldehyde, followed by H&E staining analysis. H&E staining was performed by Wuhan Saiwei Biotechnology Co., Ltd. The mouse eyeballs were removed, and venous blood was collected from the eyeballs. The detection of cytokines and type I interference color in mouse serum was the same as in Example 1.

[0047] In mice with DSS-induced colitis, RNF219-deficient mice had significantly higher body weight, stool characteristics scores, and bloody stool scores during the course of colitis compared to wild-type mice. Figure 5(A). In mice treated with normal drinking water, the colon lengths of wild-type and RNF219-deficient mice were roughly equivalent, but after DSS-induced colitis, the colon length of RNF219-deficient mice was significantly longer than that of wild-type mice. Figure 5 (B, C). H&E staining showed that wild-type mice had significantly more inflammatory cell infiltration in the intestinal tissue after induced colitis than RNF219-deficient mice. Figure 5 (D). ELISA analysis of serum from mice treated with normal drinking water revealed no significant difference in the levels of IFNβ, TNF, and IL-6 between wild-type and RNF219-deficient mice. However, after DSS-induced colitis, the levels of inflammatory cytokines and type I interferon in the serum of RNF219-deficient mice were significantly lower than those in wild-type mice. Figure 5 (E).

[0048] These results demonstrate that RNF219 deficiency can delay the progression of colitis in mice and improve the inflammatory phenotype of the colon in mice.

[0049] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. The application of substances that inhibit the activity of RNF219 protein or reduce the content of RNF219 protein in the preparation of drugs for the prevention or treatment of inflammatory diseases.

2. The application according to claim 1, characterized in that, The inflammatory diseases include sepsis and colitis, preferably, colitis is inflammatory bowel disease.

3. The application according to claim 1, characterized in that, The substance that inhibits the activity of RNF219 protein is a protein, polypeptide, or small molecule compound that inhibits the function of RNF219 protein; or, the substance that reduces the content of RNF219 protein is a substance that inhibits the synthesis of RNF219 protein, promotes the degradation of RNF219 protein, or knocks down or eliminates the RNF219 gene.

4. The application according to claim 3, characterized in that, The substance that inhibits the activity of RNF219 protein includes RNF219 antibody.

5. The application according to claim 4, characterized in that, The substance that inhibits RNF219 protein synthesis includes a substance that inhibits the expression of the gene encoding RNF219 protein, preferably a substance that silences the gene encoding RNF219 protein.

6. The application according to claim 5, characterized in that, The substance that knocks down or eliminates the RNF219 gene includes the RNF219 gene knockout system.

7. A drug for the prevention or treatment of inflammatory diseases, characterized in that, The active ingredient of the drug is a substance that inhibits the activity of RNF219 protein or reduces the content of RNF219 protein.

8. The drug according to claim 5, characterized in that, The substance that inhibits the activity of RNF219 protein is a protein, polypeptide, or small molecule compound that inhibits the function of RNF219 protein; or, the substance that reduces the content of RNF219 protein is a substance that inhibits the synthesis of RNF219 protein, promotes the degradation of RNF219 protein, or knocks down or eliminates the RNF219 gene.

9. The application of RNF219 as a target in screening drugs for the prevention or treatment of inflammatory diseases, characterized in that, The application is that if a substance has the function of inhibiting RNF219 protein synthesis, promoting RNF219 protein degradation, or knocking down or eliminating the RNF219 gene, then the substance is a candidate drug for the prevention or treatment of inflammatory diseases.

10. The application according to claim 9, characterized in that, The inflammatory diseases include sepsis and colitis, preferably, colitis is inflammatory bowel disease.

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