Zebrafish animal model for inflammatory diseases 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 a method for creating a zebrafish embryo and adult model of inflammation using glycotoxic CML, which is very useful for developing drugs for preventing or treating inflammatory diseases, and a method for screening anti-inflammatory agents using the same.

Background Art

[0002] The inflammatory response, which is one type of immune response, is a body defense mechanism after exposure to harmful stimuli such as microorganisms or compounds, and refers to the entire process from the removal of the stimulus to the repair of damaged tissues. When the inflammatory response occurs chronically, inflammatory mediators are over-secreted, which has been reported to promote the growth of cancer cells or increase insulin resistance, thereby worsening arteriosclerosis and being involved in various pathological mechanisms. In the immune system, macrophages play an important role in regulating the inflammatory response, immune function, and maintaining homeostasis. When stimulated by LPS (lipopolysaccharide), macrophages become more active. LPS is a complex of lipid and polysaccharide covalently bound to an external antigen and is an endotoxin mainly present as a component of the outer membrane of Gram-negative bacteria. Activated macrophages by stimulation promote the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. When these inflammatory mediators are formed, they play a major role in mediating inflammation by being involved in the process of converting arachidonic acid into leukotrienes, thromboxanes, and prostaglandins via the action of COX (cyclooxygenase), as well as the massive production of NO (nitric oxide), thereby causing fatal damage to the host. Among these, NO, which is a free radical, is a highly reactive substance produced from L-arginine by NOS (NO synthase), and 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 produce a large amount of NO when stimulated by external stimuli or inflammatory cytokines. Excessive NO production is known to induce excessive immune system abnormalities that cause an inflammatory response.

[0003] Cytokines, which are inflammatory mediators, belong to the class of growth factors. Cytokines are small molecules released by cells into the bloodstream, enabling immune cells to move to the site of infection, phagocytize damaged cells, and even infiltrate the blood vessel wall. Cytokines act as endogenous mediators that assist in inflammatory signaling. Cytokines themselves are essential for the immune system. When foreign antigens are introduced, pro-inflammatory cytokines are released from immune cells to kill and neutralize the foreign antigens. Representative pro-inflammatory cytokines included in this process are tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interleukin-8 (IL-8), which are mainly produced by activated macrophages and induce an inflammatory response against exogenous pathogens.

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

[0005] Cytokines can cause inflammation, which can lead to swelling, fever, and pain in the damaged area. Overproduction of cytokines is lethal 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 a large amount of various cytokines, thereby rapidly increasing the levels of a number of 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 the stage where mild reactions are shown and can be treated with antipyretics. Grade 2 is the stage where weak reactions are shown within 24 hours. Grade 3 is the stage where long-term reactions are shown and the symptoms can be improved but may recur later (such as renal insufficiency, pulmonary infiltration, etc.). Grade 4 is the stage where life may be threatened and vasopressors or ventilators may be required. Grade 5 is the stage where multiple organ functions decline and are lost in a short 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 parameter, and elevated ferritin and C protein. Clinically, common causes of cytokine storm syndrome include CAR T therapy, H5N1, H1N1, SARS, MERS, and COVID-19, and the mainly included cytokines are 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 mechanism of cytokine storm syndrome is not clearly understood (possibly associated with cytolytic immune response), and there is no special therapeutic agent for treating it. Currently, most of the clinical treatment experience of cytokine storm syndrome results from immunotherapy, specifically adoptive cell therapy. Drugs that have been tried to treat cytokine storm syndrome include peroxisome proliferator-activated receptor agonist, sphingosine-1-phosphate receptor agonist, cyclooxygenase inhibitor, antioxidant, anti-tumor necrosis factor therapy, intravenous immunoglobulin, and other therapies.

[0008] Acute or excessive inflammation is associated with the cytokine cascade and is known to be closely related to sudden death and symptom exacerbation resulting from recent COVID-19 infections, pain caused by chronic inflammation, and exacerbation of autoimmune diseases (such as rheumatoid arthritis, psoriasis, sepsis, etc.). Glycation, a non-specific non-enzymatic process in which carbohydrates are linked to proteins, produces advanced glycation end products (AGEs), which in turn cause excessive inflammation, trigger cytokine storms, cause nerve paralysis, leading to a lack of motor ability.

[0009] High-density lipoprotein (HDL) in the blood is well-known to have antioxidant and anti-inflammatory effects. However, when HDL is glycated, the amount of dysfunctional HDL, which is deformed and has impaired function, increases, and this is known to actually exacerbate inflammation. Normal HDL has excellent antiviral activity and can kill the COVID-19 virus, while abnormal HDL loses its antiviral activity and shows macrophage killing, skin fibroblast killing, and embryotoxicity (Cho, K.H.; Kim, J.R.; Lee, I.C.; Kwon, H.J. 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).

[0010] Among the advanced glycation end products (AGEs), N-ε-carboxymethyllysine (CML) is known to promote LDL oxidation sensitivity in diabetic patients, thereby promoting and worsening 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 U S A 1994;91:9441-5). However, there has been no report on whether CML causes acute nerve paralysis and lack of motor ability.

[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 high-sensitivity C-reactive protein (hs-CRP), an inflammatory marker (The Journal of Clinical Endocrinology & Metabolism, 93(2), 578-583).

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

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

[0014] Moreover, cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) may be involved in neuroinflammation. Generally, neuroinflammation is associated with the onset of various neurological and neurodegenerative diseases such as cerebral ischemia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Microglia, which are innate immune cells in the brain, 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 (IL)-6, which can lead to neurotoxicity and motor neuron paralysis and contribute to neurodegenerative diseases.

[0015] Mammals, as animal models for new drug development and disease research, have the advantages of genetic and physiological similarities to humans. However, due to technical support issues, high costs, and requirements for breeding conditions in animal facilities, they are not suitable for large-scale screening. Therefore, there has been a need for alternative experimental animals with high fertility, low costs, and simple breeding requirements. Since the establishment of animal models in the early 1980s, zebrafish (Danio rerio) has been developed as an important organism for drug screening. Zebrafish has significant advantages over other vertebrate models because it is uniquely suitable for screening in 96-well or 384-well plate formats. Furthermore, the zebrafish genome is approximately 80% homologous to the human genome, which gives it an advantage over relatively small biological 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 enables more efficient research, and have transparent embryos with a short development time of 48 hours, which allows for real-time observation. In particular, zebrafish have the advantage of being able to observe specific organs in vivo in real time by expressing organ-specific biomarkers such as fluorescent proteins and can be tracked at the cellular level during embryogenesis. In addition, since zebrafish perform in vitro fertilization, it is easy to manipulate their genes by injecting genetic material into the yolk of the fertilized eggs.

Prior Art Documents

Patent Documents

[0017]

Patent Document 1

Non-Patent Documents

[0018] [Non-Patent Document 1] Cho, K.H.; Kim, J.R.; Lee, I.C.; Kwon, H.J. 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 U S A 1994;91:9441-5 [Non-Patent Document 3] The Journal of Clinical Endocrinology & Metabolism, 93(2), 578-583 [Summary of the Invention]

[0019] Utilizing these biological characteristics of zebrafish, zebrafish are widely used as an experimental model. There is a prior art document (Korean Patent No. 10-1146821) regarding the construction of a zebrafish animal model for inflammatory diseases treated with lipopolysaccharide (LPS), oxidized low density lipoprotein (oxLDL), and glycated apoA-I (gA-1) that cause chronic inflammation. However, the present invention discloses for the first time a zebrafish animal model for inflammatory diseases using carboxymethyllysine (CML). [Problems to be Solved by the Invention]

[0020] [Summary of the Invention] By injecting carboxymethyllysine (CML), an advanced glycation end product, into adult zebrafish and embryos to induce acute death and developmental disorders in embryos and nerve paralysis in adults, and by co-injecting a test substance that suppresses their effects with CML to compare the degree and rate of inflammation reduction and paralysis recovery, it is an object of the present invention to provide a useful method for screening novel anti-inflammatory agents. [Means for Solving the Problems]

[0021] To achieve the above object, the present invention provides the following steps: (1) Inducing inflammation in zebrafish by treating with CML; (2) Injecting CML together with a test substance into zebrafish; and (3) Confirming the effect of the test substance by comparing the above zebrafish with those treated with CML alone to provide a method for screening novel anti-inflammatory agents. [Advantages 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 adult zebrafish and embryos and the advanced glycation end product CML to induce embryo death, acute cytokine storm, and nerve paralysis. [Brief Description of the Drawings]

[0023]

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Mode for Carrying Out the Invention

[0024] In the present specification, the present invention will be described in detail below.

[0025] 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 having average knowledge in the relevant field that the embodiments of the present invention are provided to more accurately explain the present invention.

[0026] The present invention includes the following steps: (1) Inducing inflammation in zebrafish by treating with CML; (2) Injecting CML together with a test substance into the zebrafish; and (3) Confirming the action of the test substance by comparing the above zebrafish with those treated with CML alone. A method for screening an anti-inflammatory agent is provided.

[0027] The zebrafish in the above step (1) is characterized by its similarity to mammalian organs, and since most of those organs develop within several hours after fertilization, the time of organogenesis and the developmental stage are very rapid. Zebrafish develop very rapidly. In particular, the initial cell division of zebrafish occurs at 15-minute intervals, which is faster than that of Escherichia coli (20 minutes). Gastrulation begins at 6 hours of development, is completed at 10 hours, and eye formation begins 12 hours later. After 24 hours of fertilization, heartbeat and blood cell flow can be observed, and the major organs and systems are completely formed 5 to 6 days after fertilization.

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

[0029] The initial developmental stages of zebrafish are seven stages in the order of zygote stage, cleavage stage, blastula stage, gastrula stage, somitogenesis stage, pharyngula stage, and hatching stage.

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

[0031] The above CML belongs to advanced glycation end products (AGE).

[0032] Advanced glycation end products are formed by the Maillard reaction that occurs between amino acid groups such as lysine residues of proteins and reducing sugars without the action of enzymes. Non-enzymatic glycation of proteins 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. Subsequently, the compounds formed at this point undergo a series of complex reactions such as condensation, rearrangement, oxidation, cleavage, and cyclization, resulting in the formation of brown compounds (melanoidins), which form irreversible advanced glycation end products. Since advanced glycation end products are irreversible reaction products, once formed, they are not decomposed even when blood glucose levels return to normal. Instead, they accumulate in tissues throughout the lifespan of proteins, abnormally changing the structure and function of tissues. Collagen, which has a relatively long half-life, is easily glycated and forms cross-links with already formed advanced glycation end products, which causes abnormal physicochemical changes in the structure of the skin such as facial wrinkles and other protein-binding tissues in the body. In addition, advanced glycation end products are recognized by specific receptors for various cell types, causing diabetic complications such as diabetic retinopathy, diabetic neuropathy, diabetic cataract, and diabetic nephropathy, as well as other diseases such as diabetes, chronic kidney disease, heart disease, vascular disease, and aging.

[0033] Irreversibly formed advanced glycation end products include N ε -(1-carboxyethyl)lysine (CEL), N ε -(1-carboxymethyl)lysine (CML), methylglyoxal-derived hydroimidazolone N σ -(5-hydroxy-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1), glyoxal-derived hydroimidazolone (G-H1), argpyrimidine, glyoxal-derived lysine dimer, 1,3-di(Nε -lithionimidazole salt)(GOLD), methylglyoxal-derived lysine dimer, 1,3-di(N ε -lithino)-4-methylimidazole salt (MOLD), etc.

[0034] The CML is a major advanced glycation end product resulting from the oxidative degradation of Amadori products, is thermochemically stable, and exhibits a relatively simple structure. CML can be produced by 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 Wolf pathway in which glucose undergoes auto-oxidation to produce highly reactive intermediates such as glyoxal, which then react with proteins to form CML. The third pathway is the Nimiki pathway in which Schiff bases formed by glucose reacting with lysine residues in proteins undergo oxidative degradation to form CML without undergoing Amadori rearrangement.

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

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

[0037] The average development time of the zebrafish development stage is 0 to 3 / 4h for the zygotene stage, 3 / 4~2 1 / 4h for the cleavage stage, 2 1 / 4~5 1 / 4h for the blastula stage, 5 1It is from 4 h to 10 h, 10 to 24 h for the somite formation period, 24 to 48 h for the pharyngeal embryo period, and 48 h to 72 h for the hatching period.

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

[0039] The zygotic stage of zebrafish is the state where the egg is newly fertilized through the completion of the first zygotic cell cycle. During the cleavage stage, the egg divides through six cycles of the 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage, and 64-cell stage. The blastula stage starts when 128 cells are visible, and the cells replicate every 15 minutes, reaching about 1000 cells 3 hours after fertilization. Four hours after fertilization, the embryo forms a sphere, and a dome shape can be observed inside. During the gastrula stage, a neural plate representing the primitive brain is formed, and there are cells that form the notochord, axial somite-derived muscles, and specific neurons in the hindbrain.

[0040] The survival rate of the zebrafish embryo induced with inflammation by the above step (1) can be 14% to 32%, 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] When the survival rate of the zebrafish embryo increases 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 extension, eye pigmentation, and the presence or absence of the midbrain-hindbrain boundary (MHB) in the brain.

[0044] Since the zebrafish embryo remained at the 25-somite stage or below after CML injection, the tail did not elongate, the pigmentation of the eyes was unclear, and the midbrain-hindbrain boundary (MHB) of the brain was not observed, it can be determined that malformations were induced.

[0045] In addition, the zebrafish embryos co-injected with the test substance and CML were at stages from primordium-3 to primordium-8 after injection, showed more than 30 somites, had black pigmentation in the eyes, and had an obvious midbrain-hindbrain boundary (MHB), indicating normal development.

[0046] More preferably, the zebrafish embryos injected with each of the test substances in combination with CML were at stages from primordium-4 to primordium-6 after injection, showed more than 32 somites, had black pigmentation in the eyes, and had an obvious midbrain-hindbrain boundary (MHB), so it can be determined that they showed normal development.

[0047] The midbrain-hindbrain boundary (MHB) is highly conserved and lies within the embryonic brain of vertebrates. In zebrafish, the midbrain-hindbrain boundary is formed soon after neural tube closure and is accompanied by ventricular expansion. The midbrain-hindbrain boundary is formed by the bending of the basal surface of the neuroepithelium. The zebrafish midbrain-hindbrain boundary is formed in two steps, the first of which is to reduce the midbrain-hindbrain boundary cells to about 75% of the length of the surrounding cells. The second is the basal constriction and apical proliferation of a small group of cells contributing to the midbrain-hindbrain boundary. Even in the absence of ventricular expansion, the basal constriction still occurs, so that the midbrain-hindbrain boundary is not formed as a passive result of ventricular expansion.

[0048] The somitogenesis stage of zebrafish is the stage where somites, pharyngeal arch primordia, and ganglia develop, the tail appears, and development begins from the one-somite stage. The total number of somites formed is variable within the range of 30 - 34 somites. The name of the pharyngeal embryonic stage including the primordium stage is derived from the formation of the pharyngeal arches that give rise to the mandible and hyoid. During this period, the elongation of the zebrafish embryo decelerates, the head is compressed, fins are formed, cell pigmentation occurs, and finally the circulatory system is formed and the heart begins to beat. 24 hours after fertilization, the primordium stage 5 (24h) progresses, followed by primordium stage 15 (30h) and primordium stage 25 (36h). The hatching stage is the final stage of embryonic development, during which organ morphogenesis is almost complete and the cartilage of the head and pectoral fins has developed.

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

[0050] Adult zebrafish injected with CML may 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 the group treated with the test substance and the untreated group by staining the cells. When the infiltration of inflammatory cells in the group treated with the test substance is reduced, the test substance can be selected as an anti-inflammatory agent.

[0052] The staining of zebrafish cells can be performed using hematoxylin-eosin staining, but is not limited thereto, for observing the infiltration of inflammatory cells.

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

[0054] If the survival rate of adult zebrafish treated with a test substance increases compared to the 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 the control group not treated with the test substance and the group treated with the test substance. If the swimming ability of the group treated with the test substance increases 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, triglyceride (neutral fat), serum amyloid A, IL-6, and TNF-α, and can be used to select an anti-inflammatory agent, but is not always limited thereto.

[0057] If the levels of total cholesterol and triglyceride 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.

[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] In this specification, hereinafter, the present invention will be described in detail by the following examples.

[0060] However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited thereto.

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 zebrafish embryo animal model microinjected with CML To establish an acute cytokine storm induction system, the inventors constructed a zebrafish embryo animal model microinjected with CML. After microscopically confirming that newly spawned zebrafish embryos were 30 minutes after fertilization (0.5 hours post-fertilization (0.5 hpf)), fertilized embryos that were dividing normally were selected. A capillary glass tube (TW100F-4, World Precision Instruments, Sarasota, FL, USA) was inserted into a micro glass needle maker (PC-10, Narishige, Tokyo, Japan), the coil was adjusted to the center of the capillary glass tube, and then the needle was made at a temperature of 71.3 °C. The tip of the needle was sharpened by cutting obliquely using sharp forceps, a hole was made, and 5 μL of the injection cocktail solution was added to the micro glass needle.

[0062] To facilitate rapid microinjection, the eggs were densely packed and arranged at an appropriate distance on a 1% agarose gel with grooves at appropriate intervals. The 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 of it, and the contents were injected. The size of the formed bubble was measured, calibrated to the desired volume, and then injected into zebrafish embryos at the 2-cell stage and 4-cell stage.

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

[0064] <1-2> Comparison of the survival rates of zebrafish embryos by CML microinjection To compare the survival rates of zebrafish embryos, the inventors prepared a non-injected group (without injection), a PBS-injected control group (PBS control), and a CML-injected group. In the CML-injected group, 20 nL of a CML solution (25 mg / mL in PBS), that is, a final of 500 ng of CML, was microinjected into zebrafish embryos, and the changes in the survival rates, development rates, and development forms of each group were observed 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 the acute embryotoxicity caused by CML, which is an advanced glycation end product (Figure 1). Therefore, it was confirmed that CML microinjected into zebrafish embryos caused excessive inflammation, acute embryotoxicity, and an acute cytokine storm.

[0066] <1-3> Comparison of embryo development by CML microinjection In the case of the PBS control group, 25 hours after PBS injection, the six stages of pharyngeal embryo stage primordia, which are the normal development rate and form seen in the non-injected control group, were shown. However, the CML-injected group showed a significantly slower development rate, showed abnormal development 24 hours after CML injection, and was accompanied by individuals that remained at the 21-somite stage 25 hours after CML injection (Figure 2).

[0067] The PBS control group showed the same morphology as primordial 6 at approximately 25 hours post-fertilization, as seen in the non-injected control group (without injection). In the PBS control group and the non-injected control group, more than approximately 34 somites were observed, and the eyes darkened to black due to eye pigmentation (red arrow). Additionally, in the PBS control group, the midbrain-hindbrain boundary (MHB) was clearly visible (red solid line). On the other hand, the experimental group injected with CML showed a developmental state at approximately 19.5 hours post-fertilization (21 somites), indicating that the experimental group had approximately 21 somites. In particular, development was slow, and developmental abnormalities were present in the head and tail (blue arrowheads), eye pigmentation was very unclear, the tail did not elongate, and the MHB of the brain was not observed.

[0068] When comparing the developmental stage (21 somites), tail elongation, and the presence or absence of the brain MHB of zebrafish embryos between the non-injected group and the PBS-injected control group, in the case of the CML-injected group, it was confirmed that normal development of the embryos was not achieved, and microinjection of CML into zebrafish embryos induced abnormalities.

[0069] <1-4> Comparison of embryo survival rates by co-microinjection of CML with Remicade (infliximab) and Actemra (tocilizumab) The inventors used two biopharmaceuticals, Remicade (infliximab) and Actemra (tocilizumab), to select a new drug to suppress the acute cytokine storm caused by CML and restore nerve paralysis.

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

[0071] After 24 hours, the survival rate of embryos injected with CML and Remicade (infliximab) simultaneously was 38.4%, and the survival rate of embryos injected with CML and Actemra (tocilizumab) simultaneously was 53.1%. Compared with the PBS injection control group showing a survival rate of 67.2%, it was confirmed that the survival rate of embryos injected with CML and Actemra (tocilizumab) simultaneously was higher than that of embryos injected with CML and Remicade (infliximab) simultaneously (Figure 3).

[0072] <1-5> Comparison of embryonic development by co-microinjection of CML with Remicade (infliximab) and Actemra (tocilizumab) The PBS control group showed a normal development rate and morphology. At 25 hours after injection, it showed the midbrain-hindbrain boundary (MHB), eye pigmentation, the number of somites, and tail elongation typical of the normal development stage at about 25 - 28 hours after fertilization. Embryos co-injected with CML and Remicade (infliximab) and embryos co-injected with CML and Actemra (tocilizumab) showed approximately the same development rate as the embryos injected with PBS and did not show developmental abnormalities (Figure 4).

[0073] In the PBS control group, characteristics that appear at the development stage (primordium 6) at about 25 hours after fertilization 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 the eye cells, and the midbrain-hindbrain boundary (MHB) were clearly visible, and the tail extended normally. In the experimental groups microinjected with a mixture of CML and Remicade, or CML and Actemra, more than about 32 somites were shown, showing approximately the same development rate and characteristics as those of the PBS control group, and no developmental abnormalities were observed (Figure 4).

[0074] When CML and Remicade (infliximab), or CML and Actemra (tocilizumab) were co - microinjected into zebrafish embryos, the developmental abnormalities observed when CML was injected alone were not observed. Therefore, it was confirmed that the biopharmaceuticals used as test substances reduced the inflammatory response and cytokine storm caused by CML, thereby increasing the survival rate 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 The inventors constructed an adult zebrafish animal model injected with CML to establish an acute cytokine storm induction system.

[0076] The movements of 10 adult zebrafish (16 ± 3 weeks old) intraperitoneally injected with a CML (250 μg, 10 μL) solution (final concentration of CML: 3 mM) and 10 adult zebrafish injected with PBS were observed 30 minutes and 1 hour after injection. The zebrafish in the PBS - injected group were seen to swim actively up and down in the aquarium 30 minutes and 1 hour after injection, and it was confirmed that all zebrafish were swimming (Figures 5 and 6). On the other hand, all zebrafish in the CML - injected group did not move 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 not as active as the zebrafish injected with PBS, and the remaining zebrafish showed acute paralysis and neurotoxicity and lost their swimming ability 30 minutes and 1 hour after injection (Figures 5 and 6).

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

[0078] <2-2> Comparison of Acute Cytokine Storm Induction and Therapeutic Effects in Liver Tissue To compare the degree of acute cytokine storm induction and reduction by CML, hematoxylin-eosin (H&E) staining was performed on adult zebrafish in the PBS injection group and the CML injection group, and the degree of inflammatory cell infiltration in liver tissue was investigated.

[0079] The hematoxylin-eosin staining results showed that the liver tissue of the CML injection group was relatively dark purple and red and had more inflammatory cell infiltration compared to the liver tissue of the PBS injection group (Figure 7). Less than 16% cell infiltration was observed in the group injected with only PBS, while 27% inflammatory cell infiltration was observed in the group injected with only CML, indicating an increase in inflammatory cell activation (Figure 8).

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

[0081] Therefore, it was confirmed that injection of CML into adult zebrafish induced acute inflammation and acute cytokine storm and decreased the survival rate.

[0082] <2-3> Comparison of Therapeutic Effects of Test Substances on Acute Cytokine Storm and Neurotoxicity Caused by CML In order to compare the effects of suppressing the acute cytokine storm caused by CML and treating neurotoxicity, as test substances, two biopharmaceuticals, Remicade (infliximab) and Actemra (tocilizumab), which are used as therapeutic agents for rheumatoid arthritis, were used.

[0083] The swimming ability of zebrafish in groups injected with PBS, CML, CML and Remicade (infliximab), and CML and Actemra (tocilizumab) via intraperitoneal administration was confirmed to compare the cytokine storm inhibitory ability of the test substances (Figure 9). All zebrafish in the PBS injection group were confirmed to be swimming at the upper part of the aquarium 30 minutes and 1 hour after injection. On the other hand, zebrafish in the CML group injected with 3 mM CML could not swim 30 minutes after injection and were found lying on the bottom of the aquarium, and 2-3 zebrafish showed recovery of swimming ability 1 hour after injection. 30 minutes after injection, more zebrafish in the CML and Remicade (infliximab) injection group and the CML and Actemra (tocilizumab) injection group were swimming than in the CML injection group, and more zebrafish regained the ability to swim 1 hour after injection, which confirmed that the test substances suppressed inflammation and reduced acute paralysis and neurotoxicity.

[0084] The effects of the test substances in the CML and Remicade (infliximab) injection group and the CML and Actemra (tocilizumab) injection group were compared. As a result, when 44 μg of Actemra (tocilizumab) was injected, the recovery of swimming ability was better and the survival rate increased compared to when 43 μg of Remicade (infliximab) was injected, which indicated an improvement in acute neurotoxicity in zebrafish that could not move, were lying on the bottom, and had lost their swimming ability due to intraperitoneal administration of CML (Figures 10 and 11).

[0085] Zebrafish in the combination treatment group with Actemra (tocilizumab), an interleukin-6 (IL-6) inhibitor, showed high survival rate (about 97%) and improvement in cytokine storm. However, zebrafish in the combination treatment group with Remicade (infliximab), a tumor necrosis factor-α (TNF-α) inhibitor, showed a survival rate of 63%, which was similar to the 60% survival rate in the PBS treatment group, indicating that its effect in suppressing the acute cytokine storm and glycotoxicity caused by CML was minimal (Figure 11). Therefore, it was confirmed that the combination treatment with Actemra (tocilizumab) was more effective than the combination treatment with Remicade (infliximab) in reducing acute paralysis and neurotoxicity and restoring swimming ability.

[0086] <2-4> Comparison of the ameliorating effects of antirheumatic drugs on acute inflammation in liver tissue When comparing the infiltration of inflammatory cells using hematoxylin-eosin (H&E) staining, the CML alone treatment group and the Remicade co-treatment group showed a similarly high degree of inflammatory cell infiltration of about 23% (Figures 12 and 13). However, in the Actemra (tocilizumab) co-treatment group, the degree of inflammatory cell infiltration was reduced compared to the CML alone treatment group, indicating a significant anti-inflammatory effect and showing an inflammatory cell infiltration of about 15 - 17%, which was similar to the PBS alone treatment group. That is, the inhibitory effect of the IL-6 inhibitor Actemra (tocilizumab) on the inflammation caused by CML-induced glycotoxicity was stronger than that of the TNF-α inhibitor Remicade (infliximab).

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

[0088] Blood was collected from zebrafish in each group, plasma was isolated, and total cholesterol (TC) and triglycerides (TG) were analyzed. As a result, the CML-alone treatment group showed the highest TC and TG concentrations (Figs. 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, K. R., & Grunfeld, C. (2022). The Effect of Inflammation and Infection on Lipids and Lipoproteins. In K. R. Feingold (Eds.) et. al., Endotext. MDText.com, Inc).

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

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

[0091] As a result, serum amyloid A (SAA) was highest in the CML-alone treatment group at 59.7 ± 1.6 ng / mL, second highest in the Remicade (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-alone treatment group at 76.4 ± 6.1 pg / mL, second highest in the Remicade (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-alone treatment group at 43.5 ± 0.4 ng / mL, second highest in the Remicade (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 the cytokine storm, which correlated well with the reduction in inflammatory cell infiltration in liver tissue. However, the Remicade (infliximab) co-treatment group showed SAA, IL-6, and TNF-α concentrations similar to those of the CML-alone treatment group. This is interpreted in relation to a minimal effect on the improvement of the cytokine storm (Figures 16 - 18).

Claims

1. The following steps: (1) Inducing inflammation in zebrafish by treating with CML; (2) Injecting CML together with a test substance into the zebrafish; and (3) Confirming the effect of the test substance by comparing the above zebrafish with those treated with CML alone A method for screening an anti-inflammatory agent, comprising the steps of:

2. The method for screening an anti-inflammatory agent according to claim 1, wherein the zebrafish in step (1) is an embryo.

3. The method for screening an anti-inflammatory agent according to claim 2, wherein CML microinjected into the zebrafish embryo causes excessive inflammation, acute embryo toxicity, and acute cytokine storm.

4. The zebrafish embryo in step (1) is 15 to 45 minutes after fertilization ( 1 / 4 h to 3 / 4 h hpf (hours post-fertilization)), a method for screening an anti-inflammatory agent according to claim 1.

5. The method for screening an anti-inflammatory agent according to claim 1, wherein the zebrafish embryo in step (1) is in the 2-cell stage to 16-cell stage.

6. The method for screening an anti-inflammatory agent according to claim 1, wherein the survival rate of the zebrafish embryo caused to have inflammation in step (1) is 14% to 32%.

7. The method for screening an anti-inflammatory agent according to claim 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 an anti-inflammatory agent according to claim 1, wherein in step (3), when the survival rate of the zebrafish embryo in the group treated with the test substance is increased compared to that of the zebrafish embryo in the untreated control group, the test substance is selected as an anti-inflammatory agent.

9. The method for screening an anti-inflammatory agent according to claim 1, wherein in order to confirm that the embryo has developed normally, the developmental stage of the zebrafish, the degree of tail extension, eye pigmentation, and the presence or absence of the midbrain-hindbrain boundary (MHB) in the brain are confirmed in step (3).

10. The method for screening an anti-inflammatory agent according to claim 9, wherein when the zebrafish embryo remains at the 25-somite stage or less after CML injection, the tail does not extend, the eye pigmentation is unclear, and the midbrain-hindbrain boundary (MHB) of the brain is not observed, it is considered that malformations have been induced.

11. A method for screening an anti-inflammatory agent according to claim 9, wherein a zebrafish embryo injected with a test substance simultaneously with CML is at stages of primordial 3 to primordial 8 after injection, shows more than 30 somites, has black pigmentation visible to the eye, and shows a distinct midbrain-hindbrain boundary (MHB) in the brain, and is considered to show normal development.

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

13. The method for screening an anti-inflammatory agent according to claim 1, wherein CML injected into adult zebrafish causes an acute cytokine storm, acute paralysis, and neurotoxicity.

14. By staining cells in step (3) above, the infiltration of inflammatory cells in the group treated with the test substance and the untreated group is compared, and when 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. The method for screening an anti-inflammatory agent according to claim 1.

15. The method for screening an anti-inflammatory agent according to claim 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 an anti-inflammatory agent according to claim 15, wherein when the survival rate of adult zebrafish in the group treated with the test substance is increased compared to that of the untreated control group of adult zebrafish, the test substance is selected as an anti-inflammatory agent.

17. In step (3) above, by 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, when 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. The method for screening an anti-inflammatory agent according to claim 1.

18. The method for screening an anti-inflammatory agent according to claim 1, wherein in order to select an anti-inflammatory agent, the levels of total cholesterol, triglyceride, serum amyloid A, IL-6, and TNF-α indicating the action of the test substance in step (3) are confirmed.

19. A method for screening the anti-inflammatory agent according to claim 18, wherein the test substance is selected as an anti-inflammatory agent when the group treated simultaneously with the test substance shows lower levels of total cholesterol and triglycerides compared to a control group not treated with the test substance.

20. A method for screening the anti-inflammatory agent according to claim 18, wherein the test substance is selected as an anti-inflammatory agent when the group treated simultaneously 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 an anti-inflammatory agent, which is constructed by treating the zebrafish according to claim 1 with CML.

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