Zebrafish animal model for inflammatory disease and method for screening anti-inflammatory agents using the same

Abstract

The present invention relates to a method for creating zebrafish embryo and adult models of inflammation using glycotoxic CML, and a method for screening anti-inflammatory agents using the same. Specifically, the present invention provides a method for screening novel anti-inflammatory agents capable of preventing or treating inflammatory diseases in a simpler and more economical manner using zebrafish in which embryonic death, acute cytokine storm, and paralysis are induced.

Description

[Technical Field]

[0001] The present invention relates to methods for generating zebrafish embryonic and adult models of inflammation using glucotoxic CML, which are highly useful for developing drugs to prevent or treat inflammatory diseases, and methods for using same to screen anti-inflammatory agents. [Background technology]

[0002] The inflammatory response, a type of immune response, is the body's defense mechanism after exposure to harmful stimuli such as microorganisms or chemical compounds. It refers to the entire process from the removal of the stimulus to the repair of damaged tissue. Chronic inflammatory responses have been reported to promote cancer cell proliferation or increase insulin resistance, thereby worsening atherosclerosis and contributing to various pathological mechanisms. In the immune system, macrophages play an important role in regulating inflammatory responses and immune function and maintaining homeostasis. Macrophages become more active when stimulated by lipopolysaccharide (LPS). LPS is a lipid-polysaccharide complex covalently bound to an external antigen and is an endotoxin found primarily as a component of the outer membrane of Gram-negative bacteria. Activated macrophages promote the secretion of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. These inflammatory mediators play a key role in mediating inflammation through the conversion of arachidonic acid to leukotrienes, thromboxanes, and prostaglandins via the action of COX (cyclooxygenase) and the massive production of NO (nitric oxide), thereby causing fatal damage to the host. Among these, the free radical NO is a highly reactive substance produced from L-arginine by NOS (NO synthase). NOS is divided into two groups: cNOS (constitutive NOS) and iNOS (inducible NOS). In particular, iNOS has been reported to be expressed by various cells and to produce large amounts of NO when stimulated by external stimuli or inflammatory cytokines. Excessive NO production is known to induce immune system abnormalities, which result in an inflammatory response.

[0003] Cytokines, a class of inflammatory mediators, are growth factors. Cytokines are small molecules released by cells into the bloodstream, enabling immune cells to migrate to sites of infection, phagocytose damaged cells, and even infiltrate blood vessel walls. Cytokines act as endogenous mediators that support inflammatory signaling. Cytokines themselves are essential for the immune system, and when foreign antigens are introduced, pro-inflammatory cytokines are released from immune cells to kill and neutralize the foreign antigens. Representative pro-inflammatory cytokines include tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interleukin-8 (IL-8), which are primarily produced by activated macrophages and induce inflammatory responses against exogenous pathogens.

[0004] Inflammatory cytokines (e.g., interleukin-4 and interleukin-10) maintain the function of living organisms by halting or attenuating the progression of inflammation. The production of pro- and anti-inflammatory cytokines is tightly regulated by complex mechanisms. Imbalanced production of these two types of cytokines leads to numerous diseases, including arthritis, kidney disease, skeletal abnormalities, asthma, cancer, sepsis, neurodegeneration, neutrophilic alveolitis, hepatitis, ischemia / reperfusion, and inflammatory bowel disease.

[0005] Cytokines cause inflammation, which can lead to edema, fever, and pain in the injured area. Excessive production of cytokines is fatal to living organisms. When the immune system is overactivated or out of control due to causes such as infection, CART (chimeric antigen receptor T cell) therapy, certain drugs, or its own immune dysregulation, the immune system releases large amounts of various cytokines, which rapidly increases the levels of multiple inflammatory cytokines. This phenomenon is called cytokine storm syndrome (cytokine release syndrome: CRS). Cytokine storm syndrome is a systemic inflammatory response caused by overactivation of the immune system.

[0006] The risk of cytokine storm is internationally classified into five grades: Grade 1 is a stage where a mild response is observed and can be treated with antipyretics, Grade 2 is a stage where a weak response is observed within 24 hours, Grade 3 is a stage where a long-term response is observed and symptoms can improve but then recur (renal failure, pulmonary infiltration, etc.), Grade 4 is a stage where it can be life-threatening and vasopressors or mechanical ventilation may be required, and Grade 5 is a stage where multiple organ functions are reduced and lost for a short period of time, which can lead to death due to multiple organ failure.

[0007] Known specific symptoms of cytokine storm syndrome include fever, headache, skin rash, joint pain, muscle pain, hypotension, vascular leakage, disseminated intravascular coagulation, and even multiple organ failure. In addition to cytokine levels, related indicators of cytokine storm syndrome include lymphopenia, elevated creatinine, disturbances in coagulation parameters, and elevated ferritin and C protein. Clinically, common causes of cytokine storm syndrome include CART therapy, H5N1, H1N1, SARS, MERS, and COVID-19, and the main cytokines involved include TNF-α, IL-1, IL-2, IL-6, IL-12, IFN-α, IFN-β, IFN-γ, MCP-1, IL-8, G-CSF, MCP-1, etc. Currently, the specific pathophysiological mechanisms of cytokine storm syndrome are not clearly understood (possibly linked to a cytolytic immune response), and there are no specific therapeutic agents for its treatment. Currently, the majority of clinical experience in treating cytokine storm syndrome comes from immunotherapy, specifically adoptive cellular therapy. Drugs that have been tried to treat cytokine storm syndrome include peroxisome proliferator-activated receptor agonists, sphingosine-1-phosphate receptor agonists, cyclooxygenase inhibitors, antioxidants, anti-tumor necrosis factor therapy, intravenous immunoglobulin, and other therapies.

[0008] Acute or excessive inflammation is associated with cytokine cascades and is known to be closely related to sudden death and worsening symptoms due to recent COVID-19 infections, pain caused by chronic inflammation, and exacerbation of autoimmune diseases (rheumatoid arthritis, psoriasis, sepsis, etc.). Glycation, a nonspecific, nonenzymatic process in which carbohydrates are linked to proteins, produces advanced glycation end products (AGEs), which in turn cause excessive inflammation, trigger a cytokine storm, and cause nerve paralysis, which leads to loss of athletic ability.

[0009] High-density lipoprotein (HDL) in the blood is known to have antioxidant and anti-inflammatory properties. However, glycation of HDL increases the amount of deformed and dysfunctional HDL, which actually worsens inflammation. Normal HDL has excellent antiviral activity and can kill the COVID-19 virus, but abnormal HDL loses its antiviral activity and exhibits macrophage death, skin fibroblast death, and embryotoxicity. (Cho, KH; Kim, JR; Lee, IC; Kwon, HJ. Native high-density lipoproteins (HDL) with higher paraoxonase exert a potent antiviral effect against SARS-CoV-2 (COVID-19), while glycated HDL lost its antiviral activity. Antioxidants 2021, 10, 209).

[0010] Among advanced glycation end products (AGEs), N-ε-carboxymethyllysine (CML) is known to increase the susceptibility of LDL to oxidation in diabetic patients, thereby accelerating and exacerbating the progression of atherosclerosis (Bucala R et al. Modification of low-density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency. Proc Natl AcadSci USA 1994;91:9441-5). However, there have been no reports on whether CML causes acute nerve palsy and motor disability.

[0011] Patients with type 1 and type 2 diabetes have elevated levels of CML in their blood, which promotes the expression of toll-like receptor 4 (TLR-4) and ultimately increases the inflammatory marker high-sensitivity C-reactive protein (hs-CRP) (The Journal of Clinical Endocrinology & Metabolism, 93(2), 578-583).

[0012] These patients exhibit elevated serum levels of inflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), which are associated with the excessive inflammatory response caused by CML. This hyperinflammation leads to a cytokine storm, which may be the cause of acute inflammation and death caused by COVID-19 infection.

[0013] Known ligands of toll-like receptor 4 (TLR-4) include CML, lipopolysaccharide (LPS), and heat shock protein (HSP)-60, which are known to be elevated in diabetic patients. Diabetic patients are known to have elevated levels of not only TLR-4 but also interleukin-6 and TNF-α. Therefore, modulating toll-like receptor (TLR) signaling is known as a good method for suppressing the inflammatory cascade. It is known that the mechanism by which structural analogs of HDL suppress inflammation is through the inhibition of lipopolysaccharide (LPS), which stimulates TLR-4 inflammatory signaling. In addition, recombinant HDL has been reported to have anti-inflammatory effects by reducing TLR-4 expression and inhibiting TLR-4 inflammatory signaling.

[0014] Furthermore, cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) may be involved in neuroinflammation. Neuroinflammation is commonly associated with the development of various neurological and neurodegenerative diseases, such as cerebral ischemia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Microglia, the brain's innate immune cells, play a particularly important role in brain inflammation through the production of inflammatory cytokines, nitric oxide (NO), and other neurotoxic factors. Activated microglia can directly damage neurons by stimulating the secretion of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6, which can cause neurotoxicity and motor neuron paralysis, leading to neurodegenerative diseases.

[0015] Mammals have the advantage of genetic and physiological similarities to humans as animal models for drug development and disease research. However, they are not suitable for large-scale screening due to technical support issues, high costs, and demanding animal care requirements. Therefore, there has been a need for alternative experimental animals with high fecundity, low costs, and simple care requirements. Since their establishment as an animal model in the early 1980s, the zebrafish (Danio rerio) has been developed as an important organism for drug screening. Zebrafish have significant advantages over other vertebrate models because they are uniquely suited for screening in 96- or 384-well plate formats. Furthermore, the zebrafish genome is approximately 80% homologous to the human genome, giving it an advantage over relatively small organism models such as Drosophila melanogaster (60%) and Caenorhabditis elegans (36%).

[0016] Compared to other animal models, zebrafish are relatively inexpensive, produce a large number of fertilized eggs, which allows for more efficient research, and have transparent embryos with a short developmental time of 48 hours, which allows for real-time observation. In particular, zebrafish have the advantage that specific organs can be observed in vivo in real time by expressing organ-specific biomarkers such as fluorescent proteins, and can be tracked at the cellular level during the embryonic stage. In addition, because zebrafish undergo in vitro fertilization, their genes can be easily manipulated by injecting genetic material into the yolk of fertilized eggs. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] Korean Patent No. 10-1146821 [Non-patent literature]

[0018] [Non-Patent Document 1] Cho, KH; Kim, JR; Lee, IC; Kwon, HJ Native high-density lipoproteins (HDL) with higher paraoxonase exerts a potent antiviral effect against SARS-CoV-2 (COVID-19), while glycated HDL lost the antiviral activity. Antioxidants 2021, 10, 209 [Non-patent document 2] Bucala R et al. Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency. Proc Natl AcadSci USA 1994;91:9441-5 [Non-patent document 3] The Journal of Clinical Endocrinology & Metabolism, 93(2), 578-583 Summary of the Invention

[0019] Taking advantage of these biological characteristics of zebrafish, zebrafish are widely used as experimental models. Although there is a prior art document (Korean Patent No. 10-1146821) on the construction of a zebrafish animal model for inflammatory diseases in which lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL), and glycated apoA-I (gA-1) are treated to induce chronic inflammation, the present invention is the first to disclose a zebrafish animal model for inflammatory diseases using carboxymethyllysine (CML). [Problem to be solved by the invention]

[0020] [Summary of the Invention] It is an object of the present invention to provide a useful method for screening novel anti-inflammatory agents by injecting carboxymethyllysine (CML), an advanced glycation end product, into adult and embryonic zebrafish to induce acute death and developmental defects in embryos and neuroparalysis in adults, and by co-injecting CML with a test substance that inhibits its action to compare the extent and speed of inflammation reduction and paralysis recovery. [Means for solving the problem]

[0021] In order to achieve the above object, the present invention comprises the following steps: (1) Induce inflammation in zebrafish by treating them with CML; (2) injecting CML with a test substance into zebrafish; and (3) comparing the zebrafish treated with CML alone to confirm the effect of the test substance. The present invention provides a method for screening for novel anti-inflammatory agents, comprising: [Effects of the Invention]

[0022] The present invention provides a method for screening novel anti-inflammatory agents in a simpler and more economical manner by using zebrafish adults and embryos and the advanced glycation end product CML to induce embryonic death, acute cytokine storm, and neuroparalysis. [Brief explanation of the drawings]

[0023] [Figure 1] 1 is a graph showing survival rates after microinjection of saline (PBS) and CML into zebrafish embryos. [Figure 2]FIG. 1 shows developmental stages of zebrafish embryos 24 and 26 hours after microinjection of CML, showing areas of developmental defects and malformations in the embryos. [Figure 3] 1 is a graph comparing survival rates after co-microinjection of CML and Remsima (Infliximab), and CML and Actemra (Tocilizumab) into zebrafish embryos. [Figure 4] FIG. 1 shows the developmental stages of zebrafish embryos 24 and 26 hours after co-microinjection of CML and Remsima (infliximab), and CML and Actemra (tocilizumab) into zebrafish embryos. [Figure 5] This is a set of photographs showing that acute paralysis and neurotoxicity were induced in adult zebrafish by intraperitoneal administration of CML, and that after a certain period of time, some of the zebrafish recovered from the paralytic symptoms and regained their swimming ability. [Figure 6] 1 is a graph showing the swimming ability of zebrafish acutely paralyzed by intraperitoneal injection of CML into adult zebrafish compared to the swimming ability of zebrafish injected with PBS. [Figure 7] 1 is a set of photographs comparing the degree of inflammatory cell infiltration in liver tissue of adult zebrafish treated with PBS alone and CML alone by hematoxylin and eosin staining. [Figure 8] 1 is a graph comparing the degree of inflammatory cell infiltration in liver tissue and the rate of viability recovery in adult zebrafish treated with PBS alone and CML alone. [Figure 9] 1 is a set of photographs comparing the ability of test substances to suppress cytokine storm by simultaneously treating adult zebrafish with CML and Remsima (infliximab) and CML and Actemra (tocilizumab) and checking the swimming ability of the zebrafish. [Figure 10]1 is a graph comparing the swimming ability of zebrafish restored by the test substances by simultaneously treating adult zebrafish with CML and Remsima (infliximab), and CML and Actemra (tocilizumab), respectively, and verifying the swimming ability of the zebrafish. [Figure 11] 1 is a graph comparing the viability recovery rate of zebrafish recovered by the test substances by simultaneously treating adult zebrafish with CML and Remsima (infliximab), and CML and Actemra (tocilizumab), respectively, and checking the swimming ability of the zebrafish. [Figure 12] 1 is a set of photographs showing liver tissue comparing the degree of inflammatory cell infiltration when treated with Remsima (infliximab) and Actemra (tocilizumab) in combination with CML. [Figure 13] 1 is a graph comparing the degree of inflammatory cell infiltration when Remsima (infliximab) and Actemra (tocilizumab) are treated in combination with CML. [Figure 14] 1 is a graph comparing serum total cholesterol levels after infusion of CML alone or in combination with a test substance. [Figure 15] 1 is a graph comparing triglyceride levels after infusion of CML alone or in combination with a test substance. [Figure 16] 1 is a graph comparing serum amyloid A (SAA) levels after infusion of CML alone or in combination with a test substance. [Figure 17] 1 is a graph comparing interleukin-6 (IL-6) levels after infusion of CML alone or in combination with a test agent. [Figure 18] 1 is a graph comparing tumor necrosis factor-α (TNF-α) levels after injection of CML alone or in combination with a test agent. DETAILED DESCRIPTION OF THE INVENTION

[0024] Hereinafter, the present invention will be described in detail.

[0025] The embodiments of the present invention can be modified in various other forms, and the scope of the present invention is not limited to the following embodiments. It is well understood by those skilled in the art that the embodiments of the present invention are provided to more accurately describe the present invention.

[0026] The present invention comprises the following steps: (1) Induce inflammation in zebrafish by treating them with CML; (2) injecting CML with a test substance into zebrafish; and (3) comparing the zebrafish treated with CML alone to confirm the effect of the test substance. The present invention provides a method for screening anti-inflammatory agents, comprising:

[0027] Zebrafish, as described in step (1) above, are characterized by the similarity of their organs to those of mammals. Most of their organs develop within a few hours after fertilization, demonstrating the rapidity of organogenesis and developmental stages. Zebrafish develop very rapidly. In particular, initial cell divisions in zebrafish occur at 15-minute intervals, faster than those in E. coli (20 minutes), gastrulation begins at 6 hours of development and is completed at 10 hours, and eye formation begins at 12 hours. 24 hours after fertilization, heartbeats and blood flow can be observed, and major organs and systems are fully formed 5-6 days after fertilization.

[0028] In a specific embodiment of the present invention, the zebrafish in step (1) may be an embryo.

[0029] The early developmental stages of zebrafish are seven, in the following order: zygote, cleavage, blastula, gastrula, somitogenesis, pharyngeal embryo, and hatching.

[0030] In a specific embodiment of the present invention, a zebrafish model for screening anti-inflammatory agents can be provided by treating zebrafish with CML.

[0031] The CML belongs to the advanced glycation end products (AGEs).

[0032] Advanced glycation end products (ADGs) are formed by the Maillard reaction, which occurs without the action of enzymes, between amino acid groups, such as lysine residues, of proteins and reducing sugars. Non-enzymatic protein glycation is a reaction in which free amino groups of proteins, such as lysine or arginine, react with the carbonyl group of reducing sugars to form Schiff bases. The compounds formed at this stage then undergo a series of complex reactions, including condensation, rearrangement, oxidation, cleavage, and cyclization, to produce brown compounds (melanoidins), which form irreversible ADGs. Because ADGs are irreversible reaction products, once they are formed, they are not decomposed even when blood sugar levels return to normal. Instead, they accumulate in tissues throughout the protein's lifespan, causing abnormal changes in the structure and function of the tissues. Collagen, which has a relatively long half-life, is easily glycated and forms crosslinks with already formed ADGs, which cause abnormal physicochemical changes in the structure of skin, such as facial wrinkles, and other protein-connective tissues throughout the body. In addition, advanced glycation end products are recognized by specific receptors on various cell types and cause diabetic complications such as diabetic retinopathy, diabetic neuropathy, diabetic cataracts, diabetic nephropathy, as well as other diseases such as diabetes, chronic kidney disease, heart disease, vascular disease, and aging.

[0033] Advanced glycation end products that are irreversibly formed include N ε -(1-carboxyethyl)lysine (CEL), N ε -(1-carboxymethyl)lysine (CML), a methylglyoxal-derived hydroimidazolone N σ -(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1), glyoxal-derived hydroimidazolone (G-H1), argpyrimidine, glyoxal-derived lysine dimer, 1,3-di(Nε -lysinoimidazole salt) (GOLD), methylglyoxal-derived lysine dimer, 1,3-di(N ε -lysino)-4-methylimidazole salt (MOLD).

[0034] CML is a major advanced glycation end product resulting from the oxidative degradation of Amadori products. It is thermochemically stable and has a relatively simple structure. CML can be produced via three different reaction pathways. The first pathway is the classical Hodge pathway, in which glucose reacts with lysine residues in proteins to form Amadori compounds, which subsequently undergo oxidative degradation to form CML. The second pathway is the Wolff pathway, in which glucose undergoes autoxidation to produce highly reactive intermediates such as glyoxal, which then reacts with proteins to form CML. The third pathway is the Nimiki pathway, in which the Schiff base formed by glucose reacting with lysine residues in proteins undergoes oxidative degradation to form CML without undergoing Amadori rearrangement.

[0035] In zebrafish embryos, microinjected CML causes hyperinflammation, which can lead to acute embryotoxicity and an acute cytokine storm.

[0036] In a specific embodiment of the present invention, zebrafish embryos are embryos 15 to 45 minutes after fertilization ( 1 / 4h~ 3 The embryo may be 1 / 4 h hpf (hours post fertilization), but more preferably 25 to 35 minutes post fertilization.

[0037] The average developmental time for zebrafish developmental stages is 0 to 100 times the zygote stage. 3 / 4h, Regarding the cleavage period 3 / 4~2 1 / 4h, 2 for the blastula stage 1 / 4~5 1 / 4h, 5 for gastrula stage 1 / 4h to 10h, 10 to 24h for the somitogenesis stage, 24 to 48h for the pharyngeal embryo stage, and 48h to 72h for the hatching stage.

[0038] The zebrafish embryo may be at the 2-cell to 16-cell stage, more preferably at the 2-cell to 4-cell stage.

[0039] The zebrafish zygote stage is the state in which the egg is newly fertilized through the completion of the first zygote cell cycle. During the cleavage stage, the egg divides through six cycles: the 2-cell, 4-cell, 8-cell, 16-cell, 32-cell, and 64-cell stages. The blastula stage begins when 128 cells are observed; cells replicate every 15 minutes, reaching approximately 1,000 cells by 3 hours after fertilization. By 4 hours after fertilization, the embryo has formed a spherical shape, with an internal dome-like structure. During the gastrula stage, the neural plate, representing the primitive brain, is formed, and cells that will form the notochord, axial somite-derived muscles, and specialized neurons of the hindbrain are present.

[0040] The survival rate of zebrafish embryos in which inflammation has been induced by the above step (1) can be 14% to 32%, but is preferably 20% to 26%.

[0041] In a specific embodiment of the present invention, step (3) is a step of comparing the survival rates of zebrafish embryos in a control group not treated with a test substance and a group treated with a test substance.

[0042] If the survival rate of zebrafish embryos is increased in the group treated with the test substance compared to the control group not treated with the test substance, the test substance can be selected as an anti-inflammatory agent.

[0043] In a specific embodiment of the present invention, step (3) can confirm whether normal development of the embryo has occurred by examining the developmental stage, the degree of tail elongation, eye pigmentation, and the presence or absence of the midbrain-hindbrain boundary (MHB) in the brain.

[0044] After CML injection, the zebrafish embryos remained at the 25-somite stage or less, the tail did not elongate, the pigmentation of the eyes was unclear, and the midbrain-hindbrain boundary (MHB) was not observed in the brain, which indicates that malformations were induced.

[0045] In addition, zebrafish embryos co-injected with test substances and CML were at primordium-3 to primordium-8 stage after injection, exhibited more than 30 somites, had black pigmentation in the eyes, and had a clear midbrain-hindbrain boundary (MHB), indicating normal development.

[0046] More preferably, zebrafish embryos injected with each of the test substances in combination with CML were at anlage 4 to anlage 6 stage after injection, exhibited more than 32 somites, had black pigmentation in the eyes, and had a clear midbrain-hindbrain boundary (MHB), which can be judged to have shown normal development.

[0047] The midbrain-hindbrain boundary (MHB) is highly conserved and located throughout the vertebrate embryonic brain. In zebrafish, the midbrain-hindbrain boundary forms soon after neural tube closure and is accompanied by ventricular expansion. The midbrain-hindbrain boundary is formed by folding of the basal surface of the neuroepithelium. The zebrafish midbrain-hindbrain boundary forms in two steps, the first of which is the reduction of midbrain-hindbrain boundary cells to approximately 75% of the length of the surrounding cells. The second is the basal contraction and apical proliferation of a small group of cells that contribute to the midbrain-hindbrain boundary. Even in the absence of ventricular expansion, basal contraction still occurs, and therefore the midbrain-hindbrain boundary does not form as a passive result of ventricular expansion.

[0048] The zebrafish somitogenesis stage is the stage in which somites, pharyngeal arch primordia, and ganglia develop, and the tail appears. Development begins at the one-somite stage, with the total number of somites forming varying from 30 to 34. The name of the pharyngeal embryonic stage, including the primordium stage, comes from the formation of the pharyngeal arches that give rise to the mandibles and gills. During this period, zebrafish embryo elongation slows, the head compresses, fins form, cellular pigmentation occurs, and finally, the circulatory system forms and the heart begins beating. 24 hours after fertilization, the primordium 5 stage (24 h) progresses, followed by the primordium 15 stage (30 h) and the primordium 25 stage (36 h). The hatching stage is the final stage of embryonic development, during which organ morphogenesis is nearly complete and the cartilage of the head and pectoral fins develops.

[0049] In a specific embodiment of the present invention, the zebrafish in step (1) may be an adult.

[0050] Adult zebrafish injected with CML can exhibit acute cytokine storm, acute paralysis, and neurotoxicity.

[0051] In a specific embodiment of the present invention, step (3) is a step of comparing the infiltration of inflammatory cells in a group treated with a test substance and a group not treated by staining cells, and if the infiltration of inflammatory cells is reduced in the group treated with the test substance, the test substance can be selected as an anti-inflammatory agent.

[0052] Zebrafish cells can be stained using, but not limited to, hematoxylin-eosin staining to observe inflammatory cell infiltration.

[0053] In a specific embodiment of the present invention, step (3) can include comparing the survival rates of adult zebrafish in a control group not treated with the test substance and a group treated with the test substance.

[0054] If the survival rate of adult zebrafish treated with the test substance is increased compared to a control group not treated with the test substance, the test substance can be selected as an anti-inflammatory agent.

[0055] In a specific embodiment of the present invention, step (3) is a step of comparing the swimming ability of adult zebrafish in a control group not treated with the test substance and a group treated with the test substance, and if the swimming ability of the group treated with the test substance is increased compared to the control group, the test substance can be selected as an anti-inflammatory agent.

[0056] In a specific embodiment of the present invention, the effect of the test substance in step (3) can be determined by examining the levels of total cholesterol, triglycerides (neutral fats), serum amyloid A, IL-6, and TNF-α, and can be used to select anti-inflammatory agents, but is not necessarily limited thereto.

[0057] If the total cholesterol and triglyceride levels of the group treated with the test substance are lower than those of the control group not treated with the test substance, the test substance can be selected as an anti-inflammatory agent.

[0058] If the levels of serum amyloid A (SAA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the group treated with the test substance are lower than those in the control group not treated with the test substance, the test substance can be selected as an anti-inflammatory agent.

[0059] Hereinafter, the present invention will be described in detail by the following examples.

[0060] However, the following examples are only for the purpose of illustrating the present invention, and the contents of the present invention are not limited thereto. The present invention encompasses, for example, the following embodiments: [1]Follow these steps: (1) Induce inflammation in zebrafish by treating them with CML; (2) injecting CML with a test substance into zebrafish; and (3) comparing the zebrafish treated with CML alone to confirm the effect of the test substance. A method for screening an anti-inflammatory agent, comprising: [2] The method for screening an anti-inflammatory agent according to [1], wherein the zebrafish in step (1) is an embryo. [3] The method for screening anti-inflammatory agents described in [2], in which CML microinjected into zebrafish embryos causes excessive inflammation, acute embryotoxicity, and acute cytokine storm. [4] The zebrafish embryos in step (1) were incubated for 15 to 45 minutes after fertilization ( 1 / 4 h~ 3 / 4 The method for screening an anti-inflammatory agent according to [1], wherein the anti-inflammatory agent is a mouse model of ovarian hyperplasia (ovarian hyperplasia) in which the mouse model ... [5] The method for screening an anti-inflammatory agent according to [1], wherein the zebrafish embryos in step (1) are at the 2-cell to 16-cell stage. [6] The method for screening an anti-inflammatory agent according to [1], wherein the survival rate of zebrafish embryos induced in inflammation by the above step (1) is 14% to 32%. [7] The method for screening anti-inflammatory agents described in [1], wherein step (3) is a step of comparing the survival rates of zebrafish embryos in a control group not treated with the test substance and a group treated with the test substance. [8] The method for screening anti-inflammatory agents described in [1], wherein in step (3) above, if the survival rate of zebrafish embryos in the group treated with the test substance is increased compared to that of zebrafish embryos in the untreated control group, the test substance is selected as an anti-inflammatory agent. [9] The method for screening anti-inflammatory agents described in [1], wherein the developmental stage of the zebrafish, the degree of tail elongation, the pigmentation of the eyes, and the presence or absence of the midbrain-hindbrain boundary (MHB) in the brain are confirmed in step (3) above to confirm that the embryo has developed normally.

[10] The method for screening anti-inflammatory agents described in [9], wherein if zebrafish embryos remain at the 25-somite stage or less after CML injection, the tail does not elongate, eye pigmentation is unclear, and the midbrain-hindbrain boundary (MHB) is not observed in the brain, it is considered that malformations have been induced.

[11] A method for screening anti-inflammatory agents according to [9], in which zebrafish embryos injected with a test substance simultaneously with CML are considered to exhibit normal development if they are at the anlage 3 to anlage 8 stage after injection, exhibit 30 or more somites, have black pigmentation in the eyes, and show a clear midbrain-hindbrain boundary (MHB) in the brain.

[12] The method for screening an anti-inflammatory agent according to [1], wherein the zebrafish in step (1) is an adult.

[13] The method for screening anti-inflammatory agents described in [1], in which CML injected into adult zebrafish causes acute cytokine storm, acute paralysis, and neurotoxicity.

[14] A method for screening anti-inflammatory agents described in [1], wherein the infiltration of inflammatory cells in a group treated with a test substance and a group not treated is compared by staining the cells in step (3) above, and if the infiltration of inflammatory cells in the group treated with the test substance is reduced, the test substance is selected as an anti-inflammatory agent.

[15] The method for screening anti-inflammatory agents described in [1], wherein step (3) is a step of comparing the survival rates of adult zebrafish in a control group not treated with the test substance and a group treated with the test substance.

[16] The method for screening anti-inflammatory agents described in

[15] , wherein the test substance is selected as an anti-inflammatory agent if the survival rate of adult zebrafish in a group treated with the test substance is increased compared to that of adult zebrafish in an untreated control group.

[17] The method for screening anti-inflammatory agents described in [1], wherein in step (3) above, the swimming ability of adult zebrafish in a control group not treated with the test substance and a group treated with the test substance is compared, and if the swimming ability of adult zebrafish in the group treated with the test substance is increased compared to the untreated control group, the test substance is selected as an anti-inflammatory agent.

[18] The method for screening anti-inflammatory agents described in [1], wherein the levels of total cholesterol, triglycerides, serum amyloid A, IL-6, and TNF-α, which indicate the effect of the test substance in step (3) above, are confirmed to select an anti-inflammatory agent.

[19] The method for screening anti-inflammatory agents described in

[18] , wherein the test substance is selected as an anti-inflammatory agent if the group simultaneously treated with the test substance shows lower levels of total cholesterol and triglycerides compared to a control group not treated with the test substance.

[20] The method for screening anti-inflammatory agents described in

[18] , wherein the test substance is selected as an anti-inflammatory agent if a group simultaneously treated with the test substance shows lower levels of serum amyloid A (SAA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) compared to a control group not treated with the test substance.

[21] A zebrafish model for screening anti-inflammatory drugs constructed by treating the zebrafish described in [1] with CML. [Example]

[0061] Example 1: Establishment of an acute cytokine storm induction system in zebrafish embryos and comparison of the effects of cytokine storm inhibitors <1-1> Construction of a CML-microinjected zebrafish embryo animal model To establish an acute cytokine storm induction system, we constructed a zebrafish embryo animal model microinjected with CML. Freshly laid zebrafish embryos were microscopically confirmed to be 30 min post-fertilization (0.5 h post-fertilization (hpf)) and then selected for normal cleavage. A glass capillary tube (TW100F-4, World Precision Instruments, Sarasota, FL, USA) was inserted into a glass microneedle maker (PC-10, Narishage, Tokyo, Japan), and the coil was adjusted to be centered within the capillary tube. The needle was then prepared at 71.3°C. The tip of the needle was sharpened by cutting it diagonally with sharp forceps, creating a hole, and 5 μL of the injection cocktail solution was added to the glass microneedle.

[0062] To facilitate rapid microinjection, eggs were tightly packed and aligned at appropriate distances on a 1% agarose gel with appropriately spaced grooves. A micro-glass needle was attached to a microinjector (PV-830, World Precision Instruments, Sarasota, FL, USA), 5 μL of mineral oil was spread on a graduated hemocytometer, the needle was placed on top, and the contents were injected. The size of the formed bubble was measured and calibrated to the desired volume, which was then injected into two- and four-cell zebrafish embryos.

[0063] At this point, the microinjection was performed as far into the yolk region as possible to minimize errors during the microinjection process. After microinjection, the embryos were separated from the agarose gel and placed in a Petri dish. The Petri dish was then filled with 20 mL of egg water (0.02 mg / L methylene blue) and stored at 28°C. All injection processes were performed under a stereomicroscope (SMZ-168, Motic, Hong Kong) at 20x magnification.

[0064] <1-2> Comparison of survival rates of zebrafish embryos after CML microinjection To compare the survival rates of zebrafish embryos, we created a non-injected group (no injection), a PBS-injected control group (PBS control), and a CML-injected group. For the CML-injected group, 20 nL of CML solution (25 mg / mL in PBS) was microinjected into zebrafish embryos, resulting in a final CML dose of 500 ng. The survival rates, developmental rates, and developmental morphology of each group were monitored for 24 hours.

[0065] As a result, the non-injected group showed the highest survival rate of 88.9%, the PBS-injected control group showed a survival rate of 65.9%, and the CML-injected group showed a significantly lower survival rate of 23.5%, indicating acute embryotoxicity caused by CML, an advanced glycation end product (ADG) (Figure 1). Therefore, it was confirmed that CML microinjected into zebrafish embryos caused hyperinflammation, acute embryotoxicity, and an acute cytokine storm.

[0066] <1-3> Comparison of embryo development by CML microinjection The PBS control group showed a pharyngeal primordium stage 6 25 hours after PBS injection, which is the normal developmental rate and morphology seen in the uninjected control group. However, the CML-injected group showed a significantly slower developmental rate, with some individuals showing malformed development 24 hours after CML injection and remaining at the 21-somite stage 25 hours after CML injection (Figure 2).

[0067] The PBS control group showed the same morphology as primordium 6 at approximately 25 hours post-fertilization as the uninjected control group (no injection). Approximately 34 or more somites were observed in the PBS control and uninjected control groups, and the eyes darkened to black due to eye pigmentation (red arrows). In addition, the midbrain-hindbrain boundary (MHB) was clearly visible in the PBS control group (red solid line). On the other hand, the CML-injected experimental group showed a developmental state (21 somites) at approximately 19.5 hours post-fertilization, indicating that the experimental group had approximately 21 somites. In particular, development was slow, with developmental malformations present in the head and tail (blue arrowheads), eye pigmentation was very unclear, the tail did not elongate, and the brain MHB was not observed.

[0068] When the developmental stage (21 somites), tail elongation, and the presence or absence of brain MHB in zebrafish embryos were compared with the uninjected and PBS-injected control groups, normal embryonic development was not achieved in the CML-injected group, confirming that microinjection of CML into zebrafish embryos induced malformations.

[0069] <1-4> Comparison of embryo survival rates by combined microinjection of Remsima (infliximab) and Actemra (tocilizumab) with CML The inventors used two biopharmaceuticals, Remsima (infliximab) and Actemra (tocilizumab), to select novel drugs to suppress the acute cytokine storm caused by CML and reverse nerve paralysis.

[0070] Zebrafish embryos were microinjected with 20 nL of CML solution (25 mg / mL in PBS), i.e., a final 500 ng of CML, and co-injected with 43 ng of Remsima (infliximab), a TNF-α inhibitor, and 44 ng of Actemra (tocilizumab), an IL-6 inhibitor, respectively, and observed for changes in survival rate, developmental rate, and developmental morphology for 24 hours.

[0071] After 24 hours, the survival rate of embryos co-injected with CML and Remsima (infliximab) was 38.4%, while that of embryos co-injected with CML and Actemra (tocilizumab) was 53.1%. Compared to the PBS-injected control group, which showed a survival rate of 67.2%, the survival rate of embryos co-injected with CML and Actemra (tocilizumab) was higher than that of embryos co-injected with CML and Remsima (infliximab) (Figure 3).

[0072] <1-5> Comparison of embryo development by combined microinjection of Remsima (infliximab) and Actemra (tocilizumab) with CML The PBS control group exhibited normal developmental kinetics and morphology, with midbrain-hindbrain boundary (MHB), eye pigmentation, somite number, and tail elongation at 25 hours post-injection, typical of normal developmental stages at approximately 25-28 hours post-fertilization. Embryos co-injected with CML and Remsima (infliximab) and CML and Actemra (tocilizumab) exhibited developmental kinetics similar to that of PBS-injected embryos and showed no developmental abnormalities (Figure 4).

[0073] In the PBS control group, developmental characteristics that appear at approximately 25 hours post-fertilization (primordium 6) were observed. In the PBS control group, more than 34 somites were observed, eye pigmentation (red arrow), which is the darkening of the eyes to black due to pigmentation in eye cells, and the midbrain-hindbrain boundary (MHB) were clearly visible, and the tails were normally elongated. Experimental groups microinjected with a mixture of CML and Remsima or CML and Actemra showed approximately 32 or more somites, exhibiting developmental rates and characteristics similar to those of the PBS control group, with no developmental abnormalities observed (Figure 4).

[0074] When CML and Remsima (infliximab) or CML and Actemra (tocilizumab) were simultaneously microinjected into zebrafish embryos, the malformations observed when CML alone was injected were not observed. Therefore, it was confirmed that the biopharmaceuticals used as test substances attenuate the inflammatory response and cytokine storm caused by CML, thereby increasing survival rates and inducing normal development.

[0075] Example 2: Establishment of an acute cytokine storm induction system in adult zebrafish and comparison of the effects of cytokine storm inhibitors after paralysis induction <2-1> Construction of an adult zebrafish animal model microinjected with CML To establish an acute cytokine storm induction system, we constructed an adult zebrafish animal model injected with CML.

[0076] Ten adult zebrafish (16 ± 3 weeks old) were intraperitoneally injected with CML (250 μg, 10 μL) solution (final CML concentration: 3 mM) and ten adult zebrafish injected with PBS were observed 30 minutes and 1 hour after injection. The PBS-injected zebrafish were observed actively swimming up and down in the tank 30 minutes and 1 hour after injection, confirming that all zebrafish were swimming (Figures 5 and 6). On the other hand, all zebrafish in the CML-injected group remained motionless and lay on the bottom for at least the first 30 minutes after injection. One hour after CML injection, only 25% of the zebrafish regained their swimming ability, but they were less active than the PBS-injected zebrafish. The remaining zebrafish showed acute paralysis and neurotoxicity, losing their swimming ability 30 minutes and 1 hour after injection (Figures 5 and 6).

[0077] Therefore, it was confirmed that injection of CML into adult zebrafish induced an acute cytokine storm and acute paralysis, and exhibited neurotoxicity.

[0078] <2-2> Induction of acute cytokine storm in liver tissue and comparison of therapeutic effects To compare the degree of induction and alleviation of acute cytokine storm by CML, adult zebrafish from the PBS-injected and CML-injected groups were stained with hematoxylin and eosin (H&E) to examine the degree of inflammatory cell infiltration in the liver tissue.

[0079] Hematoxylin-eosin staining showed that the liver tissues in the CML-injected group were darker purple and red and had more inflammatory cell infiltration than those in the PBS-injected group (Figure 7). Less than 16% cellular infiltration was observed in the PBS-only injected group, whereas 27% inflammatory cell infiltration was observed in the CML-only injected group, indicating increased inflammatory cell activation (Figure 8).

[0080] When PBS was injected intraperitoneally into adult zebrafish, 100% survival was observed even after 60 minutes, whereas the survival rate after 60 minutes of injection of CML alone was less than 49%, demonstrating the extent of death caused by the acute cytokine storm (Figure 8).

[0081] Therefore, it was confirmed that injection of CML into adult zebrafish induced acute inflammation and an acute cytokine storm, and reduced survival rates.

[0082] <2-3> Comparison of the therapeutic effects of test substances on acute cytokine storm and neurotoxicity caused by CML The inventors used two biopharmaceuticals, Remsima (infliximab) and Actemra (tocilizumab), used as therapeutic agents for rheumatoid arthritis, as test substances to compare their effects on suppressing the acute cytokine storm caused by CML and treating neurotoxicity.

[0083] We compared the cytokine storm suppression abilities of test substances by examining the swimming ability of zebrafish injected intraperitoneally with PBS, CML, CML and Remsima (infliximab), and CML and Actemra (tocilizumab) (Figure 9). All zebrafish in the PBS-injected group were observed swimming at the top of the tank 30 minutes and 1 hour after injection. On the other hand, zebrafish in the CML group injected intraperitoneally with 3 mM CML were unable to swim 30 minutes after injection and were seen lying on the bottom of the tank. Only 2 to 3 zebrafish showed recovery of swimming ability 1 hour after injection. Thirty minutes after injection, more zebrafish were swimming in the CML and Remsima (infliximab)-injected group and the CML and Actemra (tocilizumab)-injected group than in the CML-injected group, and more zebrafish regained the ability to swim one hour after injection, confirming that the test substances suppressed inflammation and reduced acute paralysis and neurotoxicity.

[0084] The effects of test substances were compared between the CML and Remsima (infliximab) injection group and the CML and Actemra (tocilizumab) injection group. As a result, when 44 μg of Actemra (tocilizumab) was injected, the recovery of swimming ability was better and the survival rate was increased than when 43 μg of Remsima (infliximab) was injected, which showed an improvement in the acute neurotoxicity of zebrafish that were unable to move, lay on the bottom, and lost swimming ability due to intraperitoneal administration of CML (Figures 10 and 11).

[0085] Zebrafish treated with Actemra (tocilizumab), an interleukin-6 (IL-6) inhibitor, showed a high survival rate (approximately 97%) and improved cytokine storm, whereas zebrafish treated with Remsima (infliximab), a tumor necrosis factor-α (TNF-α) inhibitor, showed a survival rate of 63%, similar to the 60% survival rate in the PBS-treated group, indicating minimal suppression of the acute cytokine storm and glycation toxicity caused by CML (Figure 11). Therefore, Actemra (tocilizumab) treatment was more effective than Remsima (infliximab) in reducing acute paralysis and neurotoxicity and restoring swimming ability.

[0086] <2-4> Comparison of the improving effects of rheumatoid arthritis drugs on acute inflammation in liver tissue When inflammatory cell infiltration was compared using hematoxylin-eosin (H&E) staining, the CML-only treatment group and the Remsima co-treatment group showed a similarly high level of inflammatory cell infiltration, approximately 23% (Figures 12 and 13). However, in the Actemra (tocilizumab) co-treatment group, the level of inflammatory cell infiltration was reduced compared to the CML-only treatment group, indicating a significant anti-inflammatory effect, and showed an inflammatory cell infiltration of approximately 15-17%, similar to the PBS-only treatment group. In other words, the inhibitory effect of the IL-6 inhibitor Actemra (tocilizumab) on inflammation caused by CML-induced glycation toxicity was stronger than that of the TNF-α inhibitor Remsima (infliximab).

[0087] <2-5> Comparison of the effects of test substances on improving the increase in total cholesterol and triglycerides caused by CML Adult zebrafish were treated with CML at a concentration of 3 mM and simultaneously treated with Actemra (tocilizumab) and Remsima (infliximab) to confirm the effects of the test substances on reducing total cholesterol (TC) and triglycerides (TG).

[0088] Blood was collected from each group of zebrafish, plasma was isolated, and total cholesterol (TC) and triglyceride (TG) concentrations were analyzed. The CML-only treatment group showed the highest TC and TG concentrations (Figures 14 and 15). These results are consistent with previous findings that increased inflammatory cells in liver tissue increase total cholesterol and triglycerides in the blood (Feingold, KR, & Grunfeld, C. (2022). The Effect of Inflammation and Infection on Lipids and Lipoproteins. In KR Feingold (Eds.) et. al., Endotext. MDText.com, Inc.).

[0089] The PBS monotherapy group and the Actemra co-treatment group showed the lowest triglyceride levels, indicating the most significant improvement in cytokine storm, which corresponds well with the reduction in inflammatory cell infiltration in liver tissue. However, the Remsima (infliximab) co-treatment group showed similar total cholesterol and triglyceride levels to the CML monotherapy group, indicating no improvement in lipid profile. This is interpreted in relation to the minimal effect on improving cytokine storm.

[0090] <2-6> Comparison of the ameliorating effects of test substances on increases in serum amyloid A, interleukin-6, and tumor necrosis factor caused by CML Blood was collected from each group of zebrafish, and plasma was isolated and analyzed for serum amyloid A (SAA), a biomarker of hepatitis induction, interleukin-6 (IL-6), a factor that induces acute cytokine storm in the liver, and tumor necrosis factor-α (TNF-α), an inflammatory cytokine involved in liver inflammation.

[0091] As a result, serum amyloid A (SAA) was highest in the CML-only treatment group at 59.7±1.6 ng / mL, second highest in the Remsima (infliximab) treatment group at 41.2±5.3 ng / mL, and lowest in the Actemra (tocilizumab) treatment group at 38.8±2.0 ng / mL (Figure 16).

[0092] IL-6 was highest in the CML monotherapy group at 76.4±6.1 pg / mL, second highest in the Remsima (infliximab) treatment group at 62.8±3.1 pg / mL, and lowest in the Actemra (tocilizumab) treatment group at 51.8±1.8 pg / mL (Figure 17). TNF-α was highest in the CML monotherapy group at 43.5±0.4 ng / mL, second highest in the Remsima (infliximab) treatment group at 41.6±5.8 ng / mL, and lowest in the Actemra (tocilizumab) treatment group at 6.8±2.6 ng / mL (Figure 18).

[0093] The Actemra (tocilizumab) co-treatment group showed the lowest SAA, IL-6, and TNF-α concentrations, indicating the most significant improvement in cytokine storm, which corresponds well with the reduction in inflammatory cell infiltration in liver tissue. However, the Remsima (infliximab) co-treatment group showed similar SAA, IL-6, and TNF-α concentrations to the CML monotherapy group. This is interpreted in relation to the minimal effect on improving cytokine storm (Figures 16-18).

Claims

1. Steps below: (1) Induction of hyperinflammation, acute embryotoxicity, and acute cytokine storm in zebrafish embryos 15–45 min after fertilization by injecting N-ε-carboxymethyllysine (CML); (2) injecting CML together with a test substance into zebrafish embryos 15 to 45 minutes after fertilization; (3) comparing the survival rate of the zebrafish embryos of step (2) with that of the zebrafish embryos of step (1); and (4) determining that the zebrafish embryos of step (2) above are showing normal development if they are in the anlage 3 to anlage 8 stage, show more than 30 somites, have clear black pigmentation in the eyes, and show a clear midbrain-hindbrain boundary (MHB) in the brain; A method for screening an anti-inflammatory agent, comprising:

2. The method for screening anti-inflammatory agents according to claim 1, wherein the zebrafish embryos 15 to 45 minutes after fertilization are at the 2-cell to 16-cell stage.

3. The method for screening anti-inflammatory agents according to claim 1, wherein the survival rate of zebrafish embryos induced by step (1) is 14% to 32%.

4. 2. The method for screening anti-inflammatory agents according to claim 1, wherein the test substance is selected as an anti-inflammatory agent if, in step (3), the survival rate of the zebrafish embryos in the group injected with the test substance in step (2) is increased compared to that of zebrafish embryos not injected with the test substance in step (1).

5. 2. The method for screening anti-inflammatory agents according to claim 1, wherein if zebrafish embryos not injected with the test substance in step (1) remain at the 25-somite stage or less after CML injection, their tails do not elongate, their eye pigmentation is unclear, and the midbrain-hindbrain boundary (MHB) is not observed in the brain, it is considered that malformations have been induced.

6. Steps below: (1) Induce acute cytokine storm, acute paralysis, and neurotoxicity in adult zebrafish by injecting N-ε-carboxymethyllysine (CML); (2) injecting CML together with a test substance into adult zebrafish; (3) comparing the survival rate of the adult zebrafish from step (2) with that of the adult zebrafish from step (1); and (4) comparing the swimming ability of the adult zebrafish from step (1) and step (2) with that of the adult zebrafish from step (1); A method for screening an anti-inflammatory agent, comprising:

7. 7. The method for screening anti-inflammatory agents according to claim 6, wherein the infiltration of inflammatory cells in adult zebrafish in step (2) injected with the test substance and adult zebrafish in step (1) not injected with the test substance is compared by staining liver cells, and the test substance is selected as an anti-inflammatory agent if the infiltration of inflammatory cells in the adult zebrafish in step (2) is reduced.

8. 7. The method for screening anti-inflammatory agents according to claim 6, wherein in step (3), the test substance is selected as an anti-inflammatory agent if the survival rate of the adult zebrafish in step (2), which is the group injected with the test substance, is increased compared to that of the adult zebrafish in step (1), which is the untreated control group.

9. 7. The method for screening anti-inflammatory agents according to claim 6, wherein in step (4), the test substance is selected as an anti-inflammatory agent if the swimming ability of the adult zebrafish in step (2), which is the group injected with the test substance, is increased compared to the adult zebrafish in step (1), which is the untreated control group.

10. 7. The method for screening anti-inflammatory agents according to claim 6, wherein the levels of total cholesterol, triglycerides, serum amyloid A, IL-6, or TNF-α, which indicate the effect of the test substance, are confirmed in the group injected with the test substance in step (2) above, in order to select the anti-inflammatory agent.

11. 11. The method for screening anti-inflammatory agents according to claim 10, wherein the test substance is selected as an anti-inflammatory agent if the total cholesterol and triglyceride levels of the group injected with the test substance in step (2) are lower than those of the control group in step (1).

12. 11. The method for screening anti-inflammatory agents according to claim 10, wherein the test substance is selected as an anti-inflammatory agent when the levels of serum amyloid A (SAA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the group injected with the test substance in step (2) are lower than those in the control group in step (1).

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