Zebra fish animal model for inflammatory diseases and method for screening anti-inflammatory agent by using same

Abstract

The present invention relates to a method for producing zebrafish embryo and adult inflammation models using glycotoxic CML, and a method for screening anti-inflammatory agents using said models. Specifically, the present invention provides a method for screening a novel anti-inflammatory agent, which can prevent or treat inflammatory diseases in a more convenient and more economical manner, using zebrafish that has induced embryo death, acute cytokine storm, and neuroparalysis.

Description

Background Art 1. Field of the Invention

[0001] The present invention relates to a method for generating zebrafish embryonic and adult inflammation models using glucotoxic CML, which is very useful for developing drugs for preventing or treating inflammatory diseases and for screening anti-inflammatory agents using the same.

[0002] 2. Description of Related Technology

[0003] Inflammatory response is a type of immune response. It is a defense mechanism of the body after exposure to harmful stimuli such as microorganisms or compounds. It refers to the entire process from eliminating stimuli to repairing damaged tissues. It is reported that when the inflammatory response occurs for a long time, inflammatory mediators are over-secreted, promoting cancer cell growth or increasing insulin resistance, thereby aggravating arteriosclerosis and participating in a variety of pathological mechanisms. In the immune system, macrophages play an important role in regulating inflammatory responses and immune functions and maintaining homeostasis. Macrophages become more active under LPS (lipopolysaccharide) stimulation. LPS is a complex of lipids and polysaccharides that covalently link external antigens and is an endotoxin that exists mainly as a component of the outer membrane of Gram-negative bacteria. Stimulation of activated macrophages promotes the secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. It is known that when these inflammatory mediators are formed, they play a major role in mediating inflammation by participating in the conversion of arachidonic acid into leukotrienes, thromboxanes and prostaglandins under the action of COX (cyclooxygenase) and participating in the large-scale production of NO (nitric oxide), thereby causing fatal damage to the host. Among them, NO is a kind of free radical, which is a highly reactive substance produced by L-arginine through 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 in a variety of cells and produces a large amount of NO when stimulated by external stimuli or proinflammatory cytokines. It is known that excessive NO production can trigger abnormalities in the hyperimmune system that cause inflammatory reactions.

[0004] Cytokines, which are inflammatory mediators, are a class of growth factors. Cytokines are small molecules released into the blood by cells that enable immune cells to migrate to sites of infection, engulf damaged cells, and even penetrate blood vessel walls. Cytokines assist in inflammatory signaling as endogenous mediators. Cytokines themselves are vital to the immune system, and when foreign antigens are introduced, immune cells release proinflammatory cytokines to kill and neutralize the antigen. Representative proinflammatory cytokines involved at this time include tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interleukin-8 (IL-8), which are mainly produced by activated macrophages and induce inflammatory responses against exogenous pathogens.

[0005] Proinflammatory cytokines (e.g., interleukin-4 and interleukin-10) maintain the function of living organisms by preventing or attenuating the progression of inflammation. The production of proinflammatory cytokines and anti-inflammatory cytokines is strictly regulated by complex mechanisms. Imbalanced production of these two types of cytokines leads to many diseases, such as arthritis, kidney disease, bone abnormalities, asthma, cancer, sepsis, neurodegeneration, neutrophilic alveolitis, hepatitis, ischemia / reperfusion, and inflammatory bowel disease.

[0006] Cytokines cause inflammation, which can lead to swelling, fever and pain in the damaged area. Excessive production of cytokines is fatal to living organisms. When the immune system is overactivated or loses control due to reasons such as infection, CART (chimeric antigen receptor T-cell) therapy, some drugs or its autoimmune disorders, it will release a large amount of various cytokines, causing a rapid increase in the levels of a large number of pro-inflammatory cytokines. This phenomenon is called cytokine storm syndrome (CRS). Cytokine storm syndrome is a systemic inflammatory response caused by hyperactivation of the immune system.

[0007] Internationally, the risk of cytokine storm is divided into five levels. Level 1 is a stage showing mild reaction and can be treated with antipyretics, level 2 is a stage showing weak reaction within 24 hours, level 3 is a stage showing long-term reaction and symptoms can be improved but then relapse (renal failure, lung infiltration, etc.), level 4 is a stage that is life-threatening and may require pressors or mechanical ventilation, and level 5 is a stage where multiple organ functions deteriorate and are lost in the short term and death may occur due to multiple organ failure.

[0008] The specific symptoms of known cytokine storm syndrome include fever, headache, rash, arthralgia, myalgia, 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, disordered coagulation parameters, and increased ferritin and protein C. Clinically, common causes of cytokine storm syndrome include CART treatment, H5N1, H1N1, SARS, MERS, and COVID-19, and the cytokines involved mainly 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. At present, the specific pathophysiological mechanism of cytokine storm syndrome (presumably related to cytolytic immune response) is not clearly understood, and there is no specific therapeutic drug to treat it. Currently, most of the experience in the clinical treatment of cytokine storm syndrome comes from immunotherapy, especially from adoptive cell therapy. Drugs that have been tried for the treatment of 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.

[0009] Acute inflammation or excessive inflammation is associated with a cytokine cascade and is known to be closely associated with sudden death caused by recent COVID-19 infection and worsening symptoms, pain caused by chronic inflammation, and exacerbation of autoimmune diseases (rheumatoid arthritis, psoriasis, sepsis, etc.). Glycation is a non-specific, non-enzymatic process in which carbohydrates are attached to proteins, which produces advanced glycation end products (AGEs), which cause excessive inflammation, trigger a cytokine storm, and cause nerve paralysis, leading to loss of motor skills.

[0010] It is well known that high-density lipoprotein (HDL) in the blood has antioxidant and anti-inflammatory effects, but it is known that when HDL is glycated, deformed and dysfunctional HDL increases, 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 shows macrophage death, dermal fibroblast death and embryotoxicity (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).

[0011] Among advanced glycation end products (AGE), N-ε-carboxymethyl lysine (CML) is known to promote LDL oxidation sensitivity in diabetic patients, thereby accelerating the progression of arteriosclerosis and worsening it (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 Acad Sci USA 1994; 91: 9441-5). However, there is no report on whether CML causes acute nerve paralysis and motor skill loss.

[0012] The level of CML in the blood of patients with type 1 or type 2 diabetes is elevated, 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).

[0013] Increased concentrations of inflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), which are associated with excessive inflammation caused by CML, were shown in the serum of these patients. This excessive inflammation leads to a cytokine storm, which may be the cause of acute inflammation and death caused by COVID-19 infection.

[0014] Well-known ligands of Toll-like receptor 4 (TLR-4) include CML, lipopolysaccharide (LPS) and heat shock protein (HSP)-60, which are known to be increased in diabetic patients. It is well known that not only TLR-4 levels are increased in diabetic patients, but also interleukin-6 and TNF-α levels are increased. Therefore, regulating the signal transduction of Toll-like receptors (TLRs) is a well-known good way to inhibit the inflammatory cascade. It is well known that the mechanism by which HDL structural analogs inhibit inflammation inhibits lipopolysaccharide (LPS) that stimulates the inflammatory signaling of TLR-4. In addition, recombinant HDL has been reported to have anti-inflammatory effects by reducing the expression of TLR-4 and inhibiting the inflammatory signaling of TLR-4.

[0015] In addition, cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) may be associated with neuroinflammation. In general, neuroinflammation is the pathogenesis of various neurological and neurodegenerative diseases, such as cerebral ischemia, Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. Microglia are unique immune cells in the brain that play a particularly important role in brain inflammation by producing proinflammatory 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) that can cause neurotoxicity and motor neuron paralysis, thereby leading to neurodegenerative diseases.

[0016] Although mammals have the advantage of being similar to human genetics and physiology as animal models for new drug discovery and disease research, mammals are not suitable for large-scale screening due to technical support issues, high costs and demanding housing requirements in animal facilities. Therefore, alternative experimental animals with high fertility, low cost and simple housing requirements are needed. Since the early establishment of animal models in the 1980s, zebrafish (zebrafish / Danio rerio) has been developed as an important organism for drug screening. Compared with other vertebrate models, zebrafish has significant advantages because they are uniquely suitable for screening in 96-well or 384-well plate formats. In addition, the zebrafish genome is about 80% homologous to the human genome, making it superior to smaller biological models, such as Drosophila melanogaster (60%) and Caenorhabditis elegans (Caenorhabditis elegans) (36%).

[0017] Compared with other animal models, zebrafish have lower costs, produce more fertilized eggs, have high research efficiency, and have transparent embryos and a short development period of 48 hours, allowing real-time observation. In particular, zebrafish have the advantages of being able to observe specific organs in real time in vivo by expressing organ-specific biomarkers (such as fluorescent proteins), and being able to trace at the cellular level during the embryonic stage. In addition, since zebrafish are fertilized in vitro, it is easy to manipulate their genes by injecting genetic material into the yolk of fertilized eggs.

[0018] Taking advantage of these biological characteristics of zebrafish, zebrafish are widely used as experimental models. Although there are prior art documents (Korean Patent No. 10-1146821) on the construction of zebrafish animal models for inflammatory diseases, in which lipopolysaccharide (LPS), oxidized low-density lipoprotein (oxLDL) and glycosylated apoA-I (gA-1) treatments are performed to induce chronic inflammation, the present invention discloses for the first time a zebrafish animal model for inflammatory diseases using CML carboxymethyllysine (CML).

[0019] Prior art literature

[0020] Patent Literature

[0021] (Patent Document 0001) Korean Patent No. 10-1146821. Summary of the invention

[0022] The object of the present invention is to provide a useful method for screening novel anti-inflammatory agents, the method inducing acute death and developmental disorders in embryos and neural paralysis in adults by injecting carboxymethyllysine (CML), an advanced glycation end product, into zebrafish adults and embryos, and comparing the extent and speed of inflammation relief and paralysis recovery by simultaneously injecting a test substance that inhibits these effects together with CML.

[0023] In order to achieve the above object, the present invention provides a method for screening a novel anti-inflammatory agent, which comprises the following steps:

[0024] 1) Steps to induce inflammation in zebrafish by CML treatment;

[0025] 2) a step of injecting CML together with a test substance into zebrafish; and

[0026] 3) A step of confirming the effect of the test substance by comparing the above zebrafish with zebrafish treated with CML alone.

[0027] Beneficial Effects

[0028] The present invention provides a method for screening novel anti-inflammatory agents in a simpler and more economical manner by inducing embryonic death, acute cytokine storm and nerve paralysis using zebrafish adults and embryos and advanced glycation end products CML. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a graph showing the survival rate after microinjection of physiological saline (PBS) and CML into zebrafish embryos.

[0030] Figure 2 Figure 2 is a diagram showing the developmental stages of zebrafish embryos at 24 and 26 hours after microinjection of CML, illustrating areas of developmental impairment and malformation in the embryos.

[0031] Figure 3 is a graph comparing the survival rate after co-microinjection of CML and Remsima (infliximab) and CML and Actemra (tocilizumab) into zebrafish embryos.

[0032] Figure 4 are graphs showing 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.

[0033] Figure 5is a set of photographs showing that acute paralysis and neurotoxicity were induced by intraperitoneal administration of CML to adult zebrafish, and after a certain period of time, some zebrafish recovered from paralysis symptoms and regained swimming ability.

[0034] Figure 6 is a graph showing the swimming ability of zebrafish acutely paralyzed by intraperitoneal injection of CML into adult zebrafish compared with the swimming ability of zebrafish injected with PBS.

[0035] Figure 7 is a set of photographs comparing the extent of inflammatory cell infiltration in liver tissues of adult zebrafish treated with PBS alone and CML alone by hematoxylin-eosin staining.

[0036] Figure 8 It is a graph comparing the extent of inflammatory cell infiltration and the viability recovery rate in liver tissue of adult zebrafish when treated with PBS alone and CML alone.

[0037] Fig. 9 This is a set of photographs comparing the ability of test substances to suppress cytokine storm by treating adult zebrafish with CML and Remsima (infliximab) and CML and Actemra (tocilizumab) at the same time and confirming the swimming ability of the zebrafish.

[0038] Fig.10 It 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 confirming the swimming ability of the zebrafish.

[0039] Fig.11 It 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 confirming the swimming ability of the zebrafish.

[0040] Fig.12 is a set of photographs showing liver tissues comparing the extent of inflammatory cell infiltration when Remsima (infliximab) and Actemra (tocilizumab) were treated with CML, respectively.

[0041] Fig.13 is a graph comparing the extent of inflammatory cell infiltration when Remsima (infliximab) and Actemra (tocilizumab) are respectively treated together with CML.

[0042] Fig.14It is a graph comparing the serum total cholesterol levels after injection of CML alone or injection of CML together with the test substance.

[0043] Fig.15 It is a graph comparing the neutral fat levels after injection of CML alone or injection of CML and a test substance together.

[0044] Fig.16 is a graph comparing serum amyloid A (SAA) levels after injection of CML alone or CML together with a test substance.

[0045] Fig.17 is a graph comparing interleukin-6 (IL-6) levels after injection of CML alone or CML together with a test substance.

[0046] Fig.18 is a graph comparing the levels of tumor necrosis factor-α (TNF-α) after injection of CML alone or CML together with a test substance. DETAILED DESCRIPTION

[0047] Hereinafter, the present invention is described in detail.

[0048] The embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. It is easily understood by those skilled in the art that the embodiments of the present invention are provided to more accurately explain the present invention.

[0049] The present invention provides a method for screening anti-inflammatory agents, comprising the following steps:

[0050] 1) Steps to induce inflammation in zebrafish by CML treatment;

[0051] 2) a step of injecting CML together with a test substance into zebrafish; and

[0052] 3) A step of confirming the effect of the test substance by comparing the above zebrafish with zebrafish treated with CML alone.

[0053] The zebrafish in step 1) above is characterized in that its organs are similar to those of mammals, and most of its organs develop within a few hours after fertilization, so the time and development stage of organ formation are very short. Zebrafish develop very quickly. In particular, the early cell division of zebrafish is carried out at intervals of 15 minutes, which is faster than the division of Escherichia coli (20 minutes), gastrulation begins at 6 hours of development and is completed at 10 hours, and eyes begin to form after 12 hours. 24 hours after fertilization, heartbeat and blood cell flow can be observed, and 5 to 6 days after fertilization, the main organs and systems are fully formed.

[0054] In a specific embodiment of the present invention, the zebrafish in step 1) may be an embryo.

[0055] There are seven stages in the early development of zebrafish, namely the zygote stage, cleavage stage, blastocyst stage, gastrula stage, somite stage, pharyngeal pouch stage and hatching stage.

[0056] In a specific embodiment of the present invention, a zebrafish model for screening anti-inflammatory agents can be provided, wherein the model is constructed by treating zebrafish with CML.

[0057] The above-mentioned CML belongs to advanced glycation end products (AGE).

[0058] Advanced glycation end products are formed by the Maillard reaction, which occurs between amino acid groups (such as lysine residues) of proteins and reducing sugars in the absence of enzymes. Non-enzymatic glycosylation of proteins is a reaction in which free amino groups (such as lysine or arginine) of proteins react with carbonyl groups of reducing sugars to form Schiff bases, and the compounds formed then undergo a series of complex reactions such as condensation, rearrangement, oxidation, cleavage, and cyclization to produce brown compounds (melanoidine), forming irreversible advanced glycation end products. Advanced glycation end products are irreversible reaction products, so once produced, they will not degrade even when blood sugar levels return to normal, but will accumulate in tissues throughout the life of the protein, thereby abnormally changing the structure and function of the tissue. Collagen, which has a relatively long half-life, is easily glycosylated and forms cross-links with advanced glycation end products that have already been formed, causing abnormal physical and chemical changes in skin structure (such as facial wrinkles) and other protein connective tissues in the body. In addition, advanced glycation end products are recognized by specific receptors on multiple cell types and contribute to diabetic complications (e.g., diabetic retinopathy, diabetic neuropathy, diabetic cataracts, diabetic nephropathy) and other diseases (e.g., diabetes, chronic kidney disease, heart disease, vascular disease, and aging).

[0059] Irreversibly formed advanced glycation end products include Nε-(1-carboxyethyl)lysine (CEL), Nε-(1-carboxymethyl)lysine (CML), methylglyoxal-derived hydroimidazolone Nσ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1), glyoxal-derived hydroimidazolone (G-H1), arginine pyrimidine, glyoxal-derived lysine dimer, 1,3-di(Nε-lysyl imidazolium salt) (GOLD), methylglyoxal-derived lysine dimer, 1,3-di(Nε-lysyl)-4-methylimidazolium salt (MOLD), etc.

[0060] The CML is the main advanced glycation end product produced by the oxidative degradation of Amadori products. CML is thermochemically stable and has a relatively simple structure. CML can be produced by three different reaction pathways. The first approach is the classic Hodge approach, in which glucose reacts with lysine residues in proteins to form Amadori compounds, which are then oxidatively degraded to form CML. The second approach is the Wolff approach, in which glucose undergoes self-oxidation to produce highly reactive intermediates such as glyoxal, which are then reacted with proteins to form CML. The third approach is the Nimiki approach, in which the Schiff bases formed by the reaction of glucose with lysine residues in proteins are not subjected to the Amadori potential and are oxidatively degraded to form CML.

[0061] In zebrafish embryos, microinjection of CML induces excessive inflammation, leading to acute embryotoxicity and acute cytokine storm.

[0062] In a specific embodiment of the present invention, the zebrafish embryo may be an embryo at 15 to 45 minutes post fertilization (1 / 4h to 3 / 4h hpf (hours post fertilization)), but more preferably an embryo at 25 to 35 minutes post fertilization.

[0063] The average developmental time of the zebrafish developmental stages is 0-0.75h in the zygote stage, 0.75-2.25h in the cleavage stage, 2.25-5.25h in the blastocyst stage, 5.25h-10h in the gastrula stage, 10-24h in the somite stage, 24-48h in the pharyngeal pouch stage, and 48h-72h in the hatching stage.

[0064] The zebrafish embryos may be at the 2-cell stage to the 16-cell stage, but more preferably are at the 2-cell stage to the 4-cell stage.

[0065] The zygote stage of zebrafish is wherein by completing the first zygote cell cycle, the state that makes the new fertilization of ovum, and during the cleavage stage, six cycles of 2-cell stage, 4-cell stage, 8-cell stage, 16-cell stage, 32-cell stage and 64-cell stage make the fertilized egg experience cleavage.Blastocyst stage begins when there are 128 cells, and cell is replicated once in every 15 minutes, reaches about 1000 cells in 3 hours after fertilization, and 4 hours after fertilization, embryo forms spherical and can observe dome shape inside.In the gastrula stage, form the neural plate representing the original brain, and there is the cell that forms the muscle derived from notochord, axial somite and the specific neuron of hindbrain.

[0066] The survival rate of zebrafish embryos with inflammation induced by the above step 1) may be 14% to 32%, preferably 20% to 26%.

[0067] In a specific embodiment of the present invention, step 3) is a step of comparing the survival rates of zebrafish embryos of a control group not treated with the test substance and a group treated with the test substance.

[0068] If the survival rate of zebrafish embryos in the group treated with the test substance is increased compared to the control group not treated with the test substance, the test substance is selected as an anti-inflammatory agent.

[0069] In a specific embodiment of the present invention, step 3) can confirm whether the embryo develops normally by examining the developmental stage, the degree of tail elongation, the pigmentation of the eyes, and the presence of the midbrain-hindbrain boundary (MHB) in the brain.

[0070] After CML injection, the above zebrafish embryos remained at the 25-somite stage or below, the tail was not elongated, the pigmentation of the eyes was unclear, and the midbrain-hindbrain boundary (MHB) of the brain was not observed, so it was judged that malformation was induced.

[0071] Furthermore, after injection, zebrafish embryos injected with both the test substance and CML were at the primordium-3 to primordium-8 stages, exhibited more than 30 somites, had black pigmentation in the eyes, and had a clear midbrain-hindbrain boundary (MHB), indicating normal development.

[0072] More preferably, after injection, zebrafish embryos injected with each test substance and CML are at the primordium-4 to primordium-6 stage, present more than 32 somites, have black pigmentation in the eyes, and have a clear midbrain-hindbrain boundary (MHB), so that it can be judged that the embryo develops normally.

[0073] The midbrain-hindbrain boundary (MHB) is highly preserved and folded in the embryonic brain of vertebrates. The midbrain-hindbrain boundary in zebrafish is formed shortly after the closure of the neural tube, accompanied by the expansion of the ventricles. The midbrain-hindbrain boundary is formed by bending the basal surface of the neuroepithelial cells. The midbrain-hindbrain boundary of zebrafish is formed in two steps, the first step is to reduce the midbrain-hindbrain boundary cells to about 75% of the length of the surrounding cells. The second step is the basal contraction and apical expansion of a small group of cells that contribute to the midbrain-hindbrain boundary. Even in the absence of expanded ventricles, basal contraction still occurs, so that the midbrain-hindbrain boundary is not formed as a passive result of ventricle expansion.

[0074] The segmental stage of zebrafish is the stage in which the segments, pharyngeal arch primordium and ganglia develop, and the tail appears. Development begins at the 1-somite stage, and the total number of segments formed is variable, ranging from 30 to 34 segments. The stage of the pharyngeal pouch stage, which includes the primordium stage, is named after the formation of the pharyngeal arches, which produce the mandibles and gills. During this period, the elongation of the zebrafish embryo slows down, the head is compressed, the fins are formed, cytochrome deposition occurs, and finally the circulatory system is formed and the heart begins to beat. After 24 hours of fertilization, the primordium-5 stage (24h) is carried out, followed by the primordium-15 stage (30h) and the primordium-25 stage (36h). The incubation period is the last stage of embryonic development, during which organ morphogenesis is quite complete, and the cartilage of the head and pectoral fins has developed.

[0075] In a specific embodiment of the present invention, the zebrafish in step 1) may be an adult.

[0076] Adult zebrafish injected with CML exhibit acute cytokine storm, acute paralysis, and neurotoxicity.

[0077] 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 cells, 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.

[0078] Hematoxylin-eosin staining can be used to stain zebrafish cells to observe the infiltration of inflammatory cells, but is not limited thereto.

[0079] In a specific embodiment of the present invention, step 3) may include the 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.

[0080] When 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 is selected as an anti-inflammatory agent.

[0081] In a specific embodiment of the present invention, step 3) is a step of comparing the swimming ability of zebrafish adults in a control group not treated with the test substance and a group treated with the test substance, and when the swimming ability of the group treated with the test substance is increased compared to the control group, the test substance is selected as an anti-inflammatory agent.

[0082] In a specific embodiment of the present invention, the effect of the test substance in step 3) can be confirmed by examining the levels of total cholesterol, triglycerides, serum amyloid A, IL-6 and TNF-α and used to select anti-inflammatory agents, but is not always limited thereto.

[0083] When the levels of total cholesterol and triglycerides 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 is selected as an anti-inflammatory agent.

[0084] When the levels of serum amyloid A (SAA), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) 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 is selected as an anti-inflammatory agent.

[0085] Hereinafter, the present invention will be described in detail by way of the following examples.

[0086] However, the following embodiments are only used to illustrate the present invention, and the contents of the present invention are not limited thereto.

[0087] Example 1: Construction of an acute cytokine storm induction system in zebrafish embryos and inhibition of cytokine storm Comparison of the effects of

[0088] <1-1> Construction of a zebrafish embryo animal model microinjected with CML

[0089] The inventors constructed a zebrafish embryo animal model microinjected with CML to establish an acute cytokine storm induction system. The zebrafish embryos that had just laid eggs were confirmed under a microscope to be embryos 30 minutes after fertilization (0.5 hours after fertilization (0.5hpf)), and the fertilized embryos that normally divided were selected. A capillary glass tube (TW100F-4, World Precision Instruments, Sarasota, FL, USA) was inserted into a micro glass needle making machine (PC-10, Narishege, Tokyo, Japan), the coil was adjusted to the center of the capillary glass tube, and then the needle was made at 71.3°C. The tip of the needle was sharpened by cutting it diagonally with sharp tweezers to form a hole, and 5μl of the injection mixture was added to the micro glass needle.

[0090] For the convenience of rapid microinjection, eggs were densely fixed and arranged on 1% agarose gel in grooves with appropriate gaps at appropriate intervals. Micro glass needles were connected to microinjectors (PV-830, World Precision Instruments, Sarasota, FL, USA), 5 μl of mineral oil was applied to a graduated hemocytometer, the needle was placed on top, and the contents were injected. The size of the bubble formed was measured and calibrated to the required volume, and then injected into zebrafish embryos at 2-cell and 4-cell stages.

[0091] At this time, in order to minimize the error during microinjection, microinjection was performed as far as possible into the yolk region. After microinjection, the embryos were separated from the agarose gel and placed in a culture dish, which was then filled with 20 mL of egg water (0.02 mg / L methylene blue) and stored at 28° C. All injections were performed under a stereomicroscope (SMZ-168, Motic, Hong Kong) at 20× magnification.

[0092] <1-2> Comparison of zebrafish embryo survival rate by CML microinjection

[0093] In order to compare the survival rate of zebrafish embryos, the inventors prepared a non-injected group (no injection), a PBS-injected control group (PBS control) and a CML-injected group. The CML-injected group was microinjected with 20 nL of CML solution (25 mg / mL PBS solution), i.e., 500 ng CML in the end, and the survival rate, development rate and developmental morphological changes of each group were observed for 24 hours.

[0094] As a result, the non-injected group showed the highest survival rate of 88.9%, while 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%, which demonstrated that the advanced glycation end product CML caused acute embryo toxicity ( Figure 1 ). Thus, it was confirmed that CML microinjected into zebrafish embryos caused excessive inflammation, acute embryotoxicity, and an acute cytokine storm.

[0095] <1-3> Comparison of embryonic development by CML microinjection

[0096] In the case of the PBS control group, 25 hours after PBS injection, the pharyngeal pouch stage primordium-6 stage was shown, which was the normal development rate and morphology seen in the non-injected control group. However, the CML-injected group showed a significantly slower development rate, with individuals showing malformed development 24 hours after CML injection and remaining at the 21-somite stage 25 hours after CML injection ( Figure 2 ).

[0097] The PBS control group demonstrates the same form as the original base-6 of about 25h after fertilization seen in the uninjected control group (no injection). In the PBS control group and the uninjected control group, about 34 or more somites are observed, and due to eye pigmentation (red arrow), eyes darken to black. In addition, in the PBS control group, the midbrain-hindbrain boundary (MHB) is clearly visible (red solid line). On the other hand, the experimental group injected with CML shows the developmental state (21-somite) of about 19.5 hours after fertilization, indicating that they have about 21-somite. Especially, the experimental group injected with CML develops slowly, and there is developmental deformity in the head and tail (blue arrow head), and eye pigmentation is very unclear, and the tail does not extend, and the MHB of the brain is not observed.

[0098] When the developmental stage (21-somite), tail elongation, and presence or absence of brain MHB of zebrafish embryos were compared with those of the non-injected group and the PBS-injected control group, it was confirmed that normal development of the embryos could not be achieved in the case of the CML-injected group and that malformations were induced by microinjection of CML into zebrafish embryos.

[0099] <1-4> CML via combined microinjection with Remsima (infliximab) and Actemra (tocilizumab) Comparison of embryo survival rates

[0100] The present inventors used two biological drugs, Remsima (infliximab) and Actemra (tocilizumab), to select a novel drug for suppressing the acute cytokine storm caused by CML and restoring nerve paralysis.

[0101] Zebrafish embryos were microinjected with 20 nL CML solution (25 mg / mL PBS solution), i.e., a final amount of 500 ng CML, and a total of 43 ng Remsima (infliximab) (a TNF-α inhibitor) and 44 ng Actemra (tocilizumab) (an IL-6 inhibitor), respectively, and the survival rate, developmental rate, and developmental morphological changes were observed for 24 hours.

[0102] After 24 hours, the survival rate of embryos co-injected with CML and Remsima (infliximab) was 38.4%, and the survival rate of embryos co-injected with CML and Actemra (tocilizumab) was 53.1%. Compared with the PBS-injected control group, which showed a survival rate of 67.2%, it was confirmed that 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 ).

[0103] <1-5> Microscopy of CML combined with Remsima (infliximab) and CML combined with Actemra (tocilizumab) Comparison of embryonic development by injection

[0104] The PBS control group showed normal developmental rate and morphology, showing the midbrain-hindbrain boundary (MHB), eye pigmentation, somite number and tail elongation typical of normal developmental stages at about 25-28 hours after fertilization at 25 hours after injection. Embryos co-injected with CML and Remsima (infliximab) and embryos co-injected with CML and Actemra (tocilizumab) showed almost the same developmental rate as embryos injected with PBS, and had no developmental defects ( Figure 4 ).

[0105] In the PBS control group, features that appeared at the developmental stage of about 25 hours after fertilization (primordium-6) were observed. In the PBS control group, more than 34 somites were observed, eye pigmentation that darkened to black due to pigmentation of the visual cells (red arrows) and the midbrain-hindbrain boundary (MHB) were clearly visible, and the tail was elongated normally. In the experimental groups microinjected with CML and Remsima, or a mixture of CML and Actema, about 32 or more somites were shown, indicating that the development rate and features were almost the same as those of the PBS control group, and no developmental defects were observed ( Figure 4 ).

[0106] When CML and Remsima (infliximab) or CML and Actemra (tocilizumab) were microinjected into zebrafish embryos at the same time, the malformation observed when CML alone was injected was not observed. Thus, it was confirmed that the biopharmaceutical used as the test substance alleviated the inflammatory response and cytokine storm caused by CML, improved the survival rate, and induced normal development.

[0107] Example 2: Construction of an acute cytokine storm induction system in adult zebrafish and cytokine storm induction after paralysis Comparison of the effectiveness of sub-storm suppressors

[0108] <2-1> Construction of an adult zebrafish animal model of CML microinjection

[0109] The present inventors constructed an adult zebrafish animal model injected with CML to establish an acute cytokine storm induction system.

[0110] The movements of 10 zebrafish adults (16±3 weeks old) injected intraperitoneally with CML (250 μg, 10 μL) solution (final CML concentration: 3 mM) and 10 zebrafish adults injected with PBS were observed 30 minutes and 1 hour after injection. At 30 minutes and 1 hour after injection, zebrafish in the PBS injection group were observed to swim up and down actively in the water tank, and all zebrafish were swimming ( Figure 5 and 6On the other hand, all zebrafish in the CML-injected group were immobilized and lying on the bottom for at least the first 30 minutes after injection. One hour after CML injection, only 25% of the zebrafish recovered their swimming ability, but their activity was low compared with that of zebrafish injected with PBS, and the remaining zebrafish showed acute paralysis and neurotoxicity, losing their swimming ability at 30 minutes and 1 hour after injection ( Figure 5 and 6 ).

[0111] Thus, it was confirmed that injection of CML into adult zebrafish induced acute cytokine storm and acute paralysis and exhibited neurotoxicity.

[0112] <2-2> Comparison of acute cytokine storm induction and treatment effects in liver tissue

[0113] To compare the extent of acute cytokine storm induced by CML and its alleviation, hematoxylin-eosin (H&E) staining was performed on zebrafish adults in the PBS-injected and CML-injected groups, and the extent of inflammatory cell infiltration in the liver tissue was investigated.

[0114] Hematoxylin-eosin staining results showed that the liver tissue of the CML injection group was dark purple and red and had higher inflammatory cell infiltration compared with the liver tissue of the PBS injection group ( Figure 7 In the PBS-only injection group, less than 16% of cell infiltration was observed, whereas in the CML-only injection group, 27% of inflammatory cell infiltration was observed, indicating increased inflammatory cell activation ( Figure 8 ).

[0115] When PBS was injected intraperitoneally into adult zebrafish, 100% survival was still observed after 60 minutes, but the survival rate was less than 49% 60 minutes after injection of CML alone, demonstrating the extent of death caused by the acute cytokine storm ( Figure 8 ).

[0116] Thus, it was confirmed that injection of CML into adult zebrafish induced acute inflammation and acute cytokine storm, and reduced survival rate.

[0117] <2-3> Comparison of acute cytokine storm induction and treatment effects in liver tissue

[0118] The present inventors used two biological drugs, Remsima (infliximab) and Actemra (tocilizumab), as test substances to compare the effects of inhibiting acute cytokine storm caused by CML and treating neurotoxicity, the two biological drugs being used as therapeutic agents for rheumatoid arthritis.

[0119] The cytokine storm suppressive ability of the test substances was compared by confirming the swimming ability of zebrafish injected with PBS, CML, CML and Remsima (infliximab), and CML and Actemra (tocilizumab) by intraperitoneal administration ( Fig. 9 ). At 30 minutes and 1 hour after injection, all zebrafish in the PBS injection group were confirmed to be swimming above the water tank. On the other hand, it can be seen that the zebrafish in the CML group injected intraperitoneally with 3mM CML all lay at the bottom of the water tank, unable to swim at 30 minutes after injection, and 2 to 3 zebrafish showed recovery of swimming ability at 1 hour after injection. At 30 minutes after injection, more zebrafish in the CML and Remsima (infliximab) injection group and the CML and Actemra (tocilizumab) injection group were swimming compared with the CML injection group, and more zebrafish regained swimming ability at 1 hour after injection, confirming that the test substance inhibited inflammation and alleviated acute paralysis and neurotoxicity.

[0120] The effects of the test substance in the CML and Remsima (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 with the injection of 43 μg of Remsima (infliximab), indicating that the acute neurotoxicity of zebrafish that could not move, lay on the bottom and lost the ability to swim due to intraperitoneal administration of CML was improved ( Fig.10 and 11 ).

[0121] The zebrafish in the group treated with Actema (tocilizumab), an interleukin-6 (IL-6) inhibitor, showed a high survival rate (about 97%) and improvement of cytokine storm, but the zebrafish in the group treated with Remsima (infliximab), a tumor necrosis factor-α (TNF-α) inhibitor, showed a survival rate of 63%, which was similar to the survival rate of 60% in the PBS-treated group, indicating that the effect of inhibiting the acute cytokine storm and glucotoxicity caused by CML was small ( Fig.11 ). Thus, the combination treatment of Actemra (tocilizumab) was confirmed to be more effective than the combination treatment of Remsima (infliximab) in alleviating acute paralysis and neurotoxicity and restoring swimming ability.

[0122] <2-4> Comparison of the effects of rheumatoid arthritis drugs on the improvement of acute inflammation of liver tissue

[0123] When the infiltration of inflammatory cells was compared using hematoxylin-eosin (H&E) staining, the group treated with CML alone and the group treated with Remsima simultaneously showed similar high inflammatory cell infiltration of approximately 23% ( Fig.12 and13 ). However, in the group treated with Actemra (Tocilizumab) at the same time, the degree of inflammatory cell infiltration was reduced compared with the group treated with CML alone, showing a significant anti-inflammatory effect, and showed about 15-17% inflammatory cell infiltration, which was similar to the group treated with PBS alone. That is, the inhibitory effect of IL-6 inhibitor Actemra (Tocilizumab) on inflammation caused by CML-induced glucotoxicity was greater than that of TNF-α inhibitor Remsima (Infliximab).

[0124] <2-5> Comparison of the improving effects of the test substances on the increase of total cholesterol and triglycerides caused by CML

[0125] Adult zebrafish were treated with CML at a concentration of 3 mM, and Actemra (tocilizumab) and Remsima (infliximab) were also treated to confirm the effects of the test substances in reducing total cholesterol (TC) and triglycerides (TG).

[0126] Blood was collected from each group of zebrafish, plasma was separated, and total cholesterol (TC) and triglycerides (TG) were analyzed. As a result, the group treated with CML alone showed the highest TC and TG concentrations ( Fig.14 and 15 ). These results are consistent with previous findings that an increase in inflammatory cells in liver tissue increases total cholesterol and triglycerides in the blood (Feingold, KR, & Grunfeld, C. (2022). The Effect of Inflammation and Infection on Lipids and Lipoproteins. In K.R. Feingold (Eds.), et al., Endotext. MDText.com, Inc).

[0127] The group treated with PBS alone and the group treated with Actemra at the same time showed the lowest triglyceride concentration, indicating that the improvement of cytokine storm was the most significant, which was very consistent with the reduction of inflammatory cell infiltration in liver tissue. However, the group treated with Remsima (infliximab) at the same time showed total cholesterol and triglyceride concentrations similar to those of the group treated with CML alone, indicating that lipid metabolism (lipid profile) was not improved. This is interpreted as being related to the minimal effect of improving cytokine storm.

[0128] <2-6> Effects of the test substance on serum amyloid A, interleukin-6 and tumor necrosis factor α caused by CML Comparison of the improvement effect of the increase

[0129] Blood was collected from each group of zebrafish, plasma was separated, and total cholesterol (TC) and triglycerides (TG) were analyzed. Blood was collected from each group of zebrafish, plasma was separated, and 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) were analyzed.

[0130] As a result, serum amyloid A (SAA) was the highest in the group treated with CML alone, which was 59.7±1.6 ng / mL, the second highest in the group treated with Remsima (infliximab), which was 41.2±5.3 ng / mL, and the lowest in the group treated with Actemra (tocilizumab), which was 38.8±2.0 ng / mL ( Fig.16 ).

[0131] IL-6 was highest in the group treated with CML alone, at 76.4±6.1 pg / mL, second highest in the group treated with Remsima (infliximab), at 62.8±3.1 pg / mL, and lowest in the group treated with Actemra (tocilizumab), at 51.8±1.8 pg / mL ( Fig.17 TNF-α was highest in the group treated with CML alone, at 43.5±0.4 ng / mL, second highest in the group treated with Remsima (infliximab), at 41.6±5.8 ng / mL, and lowest in the group treated with Actemra (tocilizumab), at 6.8±2.6 ng / mL ( Fig.18 ).

[0132] The group treated with Actemra (tocilizumab) showed the lowest SAA, IL-6, and TNF-α concentrations, indicating the most significant improvement in cytokine storm, which is very consistent with the reduction in inflammatory cell infiltration in liver tissue. However, the group treated with Remsima (infliximab) showed SAA, IL-6, and TNF-α concentrations similar to those of the group treated with CML alone. This is interpreted as being slightly related to the improvement effect of cytokine storm ( Figures 16 to 18 ).

Claims

1. A method for screening an anti-inflammatory agent, comprising the following steps: 1) Steps to induce inflammation in zebrafish by CML treatment; 2) A step of injecting CML together with a test substance into zebrafish; as well as 3) A step of confirming the effect of the test substance by comparing the above zebrafish with zebrafish treated with CML alone.

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

3. The method for screening anti-inflammatory agents according to claim 2, wherein CML microinjected into zebrafish embryos causes excessive inflammation, acute embryotoxicity and acute cytokine storm.

4. The method for screening anti-inflammatory agents according to claim 1, wherein the zebrafish embryos of step 1) are 15 to 45 minutes post fertilization (1 / 4 h to 3 / 4 h hpf (hours post fertilization)). 5 . The method for screening anti-inflammatory agents according to claim 1 , wherein the zebrafish embryos of step 1) are at the 2-cell stage to the 16-cell stage.

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

7. The method for screening an anti-inflammatory agent according to claim 1, wherein the step 3) is a step of comparing the survival rates of zebrafish embryos of 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 according to claim 1, wherein in the above step 3), if the survival rate of zebrafish embryos in the group treated with the test substance is increased compared to the survival rate 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 according to claim 1, wherein in the above step 3), the developmental stage of zebrafish, the degree of tail elongation, eye pigmentation and the presence of the midbrain-hindbrain boundary (MHB) in the brain are confirmed to ensure that the embryo develops normally.

10. The method for screening anti-inflammatory agents according to claim 9, wherein if the zebrafish embryo remains at the 25-somite stage or below after CML injection, the tail is not elongated, the pigmentation of the eyes is not clear, and the midbrain-hindbrain boundary (MHB) of the brain is not observed, the malformation is considered to be induced.

11. The method for screening an anti-inflammatory agent according to claim 9, wherein zebrafish embryos injected with the test substance and CML simultaneously are considered to exhibit normal development if they are at the primordium-3 to primordium-8 stages after injection, present a somite stage of 30 or more, have black pigmentation in the eyes, and present a clear midbrain-hindbrain boundary (MHB) in the brain.

12. The method for screening anti-inflammatory agents according to claim 1, wherein the zebrafish in step 1) is an adult. 13 . The method for screening anti-inflammatory agents according to claim 1 , wherein the CML injected into the adult zebrafish causes acute cytokine storm, acute paralysis and neurotoxicity.

14. The method for screening anti-inflammatory agents according to claim 1, wherein when the infiltration of inflammatory cells in the group treated with the test substance and the untreated group is compared by staining cells in the above step 3), and 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 an anti-inflammatory agent according to claim 1 , wherein the step 3) is a step of comparing the survival rates of zebrafish adults of 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 the test substance is selected as an anti-inflammatory agent if the survival rate of zebrafish adults in the group treated with the test substance is increased compared to the survival rate of zebrafish adults in the untreated control group.

17. The method for screening anti-inflammatory agents according to claim 1, wherein when the swimming ability of zebrafish adults in the group treated with the test substance is increased compared with that in the untreated control group by comparing the swimming ability of zebrafish adults in the control group not treated with the test substance and the group treated with the test substance in the above step 3), the test substance is selected as an anti-inflammatory agent.

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

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

20. The method for screening an anti-inflammatory agent according to claim 18, wherein the test substance is selected as an anti-inflammatory agent when a group treated with the test substance at the same time shows lower serum amyloid A (SAA), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels compared to a control group not treated with the test substance.

21. A zebrafish model for screening anti-inflammatory agents, which is constructed by treating the zebrafish of claim 1 with CML.

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