Composition for preventing, ameliorating or treating disease caused by protein nitration comprising peptide with terminal tyrosine as effective component

Abstract

To provide a composition for preventing, ameliorating or treating a disease caused by protein nitration comprising a peptide with terminal tyrosine as an effective component.SOLUTION: A peptide with terminal tyrosine has an excellent effect of inhibiting protein nitration and also an excellent effect of preventing, ameliorating or treating disease symptoms in chronic immobilization stress-induced depression / cognitive impairment model, Alzheimer's disease model, epileptic seizure model, stroke model, type 2 diabetes model, acute renal failure model, or hyperammonemia model.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing, ameliorating or treating diseases caused by protein nitration, which comprises a peptide having a tyrosine terminal as an active ingredient. [Background technology]

[0002] Oxidative stress is known to cause various diseases by damaging cells when oxygen produced during metabolic processes acts as a free radical. In particular, when reactive oxygen species (ROS) or reactive nitrogen species (RNS) present in cells generate peroxynitrite (ONOO-) at the tyrosine residues of proteins, the proteins undergo nitration. Protein nitration is known as a secondary phenomenon accompanying the oxidation process. When a specific protein undergoes nitration, its activity is reduced or it is unable to function normally due to structural mutations, resulting in various diseases. Protein nitration has been reported to be closely related to intracellular signaling, inflammatory responses, degenerative neurological disorders, and aging. It has also recently been reported to affect asthma, diabetes, cancer, chronic stress-induced depression, type 2 diabetes, and acute kidney disease. Therefore, research into protein nitration plays a vital role biologically and clinically, and there is a need for the development of substances that inhibit protein nitration.

[0003] Meanwhile, Korean Patent Registration No. 1897400 discloses a "composition for inhibiting the activity of tyrosine decarboxylase and a method for producing a fermented food using the same," and Korean Patent Publication No. 2018-0021746 discloses a "method for tableting a nitrated aromatic compound from a nitration process," but does not mention the "composition for preventing, improving, or treating a disease caused by the nitration of a protein, which contains a peptide with tyrosine at its terminal as an active ingredient" of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention has been made in response to the above-mentioned needs, and the present inventors have provided a composition for preventing, ameliorating, or treating diseases caused by protein nitration, which comprises a peptide having a tyrosine terminal as an active ingredient. They have confirmed that the active ingredient, a peptide having a tyrosine terminal, of the present invention can inhibit protein nitration and prevent, ameliorate, or treat disease symptoms in a chronic physical restraint stress-induced depression / cognitive dysfunction model, an Alzheimer's disease model, an epileptic seizure model, a stroke model, a type 2 diabetes model, an acute renal failure model, or a hyperammonemia model, thereby completing the present invention. [Means for solving the problem]

[0005] To solve the above problems, the present invention provides a functional health food composition for preventing or ameliorating diseases caused by protein nitration, which contains a peptide having a terminal tyrosine or a food-scientifically acceptable salt thereof as an active ingredient.

[0006] The present invention also provides a pharmaceutical composition for preventing or treating diseases caused by protein nitration, which comprises a peptide having a tyrosine terminal or a pharmaceutically acceptable salt thereof as an active ingredient.

[0007] The present invention also provides a composition for inhibiting the nitration of tyrosine in a protein, which comprises, as an active ingredient, a peptide having a tyrosine at its terminus or a pharmaceutically acceptable salt thereof.

[0008] The present invention also provides a method for treating a tyrosine-terminated peptide with a protein containing a nitrated tyrosine to remove the nitro group from the nitrated tyrosine.

[0009] The present invention also provides a functional health food composition for preventing or ameliorating diseases caused by an increase in reactive oxygen species / reactive nitrogen species, which contains a peptide terminated in tyrosine or a food-scientifically acceptable salt thereof as an active ingredient.

[0010] The present invention also provides a pharmaceutical composition for preventing or treating diseases caused by an increase in reactive oxygen species / reactive nitrogen species, comprising a peptide terminated with tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient. [Effects of the Invention]

[0011] The peptide of the present invention, in which tyrosine is located at the terminus, has excellent inhibitory effects on protein nitration, reduces the nitration level of tyrosine in glutamine synthetase that increases due to stress, and increases the activity of glutamine synthetase that decreases due to stress. As a result, the peptide has excellent effects on preventing, improving, or treating disease symptoms in a chronic physical restraint stress-induced depression / cognitive dysfunction model, an Alzheimer's disease model, an epileptic seizure model, a stroke model, a type 2 diabetes model, an acute renal failure model, or a hyperammonemia model. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the process of an object recognition test (ORT) and an object location recognition test (OLT) conducted to analyze the long-term memory ability of animals fed the peptide diet of the present invention. [Figure 2] The protein nitration inhibitory effect of the peptide according to the present invention was confirmed. A is the result of Western blot analysis of nitrotyrosine protein in PFC (prefrontal cortex) tissue, and B is the result of Western blot analysis of nitrotyrosine protein in liver tissue. [Figure 3]1 shows the results of Western blot analysis of insulin receptor β and phosphorylated insulin receptor β protein in liver tissue to confirm the protein expression level regulating effect of the peptide according to the present invention. [Figure 4] The results confirm the inhibitory effect of tyrosine-terminated peptides on Cu / ZnSOD (A) and MnSOD (B) nitration. PN is peroxynitrite, which induces protein nitration. * and *** indicate a statistically significant increase in Cu / ZnSOD or MnSOD activity in the peptide-treated group compared to the PN-only group. * indicates P<0.05, and *** indicates P<0.001. [Figure 5] These results confirm the inhibitory effect of a peptide with a tyrosine terminal on glutamine synthetase nitration. PN is peroxynitrite, which induces protein nitration. ** indicates a statistically significant increase in glutamine synthetase activity in the peptide-treated group compared to the PN-only treated group, p<0.01. [Figure 6] 1 shows the results of Western blot analysis confirming the inhibitory effect of catalase nitration by a peptide having tyrosine at its terminus. [Figure 7] This is the result of confirming the inhibitory effect of nitration by measuring the refolding activity of HSP60 (heat shock protein 60) by a peptide with a tyrosine terminal. RLU means the difference in absorbance (relative light unit) measured at time 0 and time 60. [Figure 8] The results show that GS activity (A), GS expression level (B), and tyrosine nitration level (C and D) of GS were confirmed by tyrosine-glutamine peptide diet (PD) in the chronic physical restraint stress-induced group (STR). [Figure 9]The graph shows the results of examining GS activity (A), GS expression level (B), and tyrosine nitration level (C and D) in the chronic restraint stress-induced group (STR) after glutamine-tyrosine peptide diet (PD). [Figure 10] The results show that plasma corticosterone levels (A), sucrose preference (B), plasma ROS / RNS levels (C), and ROS / RNS levels in PFC tissue (D) are affected by a tyrosine-glutamine peptide diet (PD) in the chronic physical restraint stress-induced group (STR). [Figure 11] The results show that plasma corticosterone levels (A), sucrose preference (B), plasma ROS / RNS levels (C), and ROS / RNS levels in PFC tissue (D) are affected by a glutamine-tyrosine peptide diet (PD) in the chronic physical restraint stress-induced group (STR). [Figure 12] Figure 1 shows the discrimination indexes showing the results of memory analysis of animals fed a tyrosine-glutamine (YQ) or tyrosine-tryptophan (YW) peptide diet in a chronic physical restraint stress animal model. A shows the results of the object recognition test (ORT) in the 1xYQ peptide diet, B shows the results of the object recognition test (ORT) in the 2xYQ peptide diet, and C shows the results of the object location recognition test (OLT) in the 1xYQ or 1xYW peptide diet. * and ** indicate a statistically significant decrease in the object recognition function index or object location recognition function index in the stress-treated group (STR) compared to the control group (CTL). * indicates P<0.05, ** indicates P<0.01. # indicates that the material cognitive function index in the peptide diet group (1xYQ or 2xYQ) was statistically significantly increased compared to the normal diet group (ND) in the stress-treated group, p<0.05. [Figure 13]These figures show the results of the object recognition test (ORT) in an animal model of Alzheimer's disease treated with a tyrosine-glutamine (YQ) peptide diet. A shows the results for 2-month-old animals, B shows the results for 8-month-old animals, C shows the results for 2-month-old and 8-month-old animals, and D shows the results for the extent of cognitive decline over 6 months. * and ** indicate a statistically significant decrease in the object recognition index of the dementia animal model (3xTG) compared to the normal group (WT). * indicates P<0.05, ** indicates P<0.01. # indicates a statistically significant decrease in the object recognition index of the 8-month-old compared to the 2-month-old group (p<0.05). [Figure 14] This figure shows the ROS / RNS levels in an animal model of Alzheimer's disease induced by a tyrosine-glutamine (YQ) peptide diet. * indicates a statistically significant increase in ROS / RNS levels in the dementia animal model (3xTG) compared with the normal group (WT), p<0.05. # indicates a statistically significant decrease in ROS / RNS levels in the peptide diet group (YQ) compared with the normal diet group (ND), p<0.05. DCF is 2',7'-dichlorofluorescein. [Figure 15]This figure shows a glutamatergic neuron fluorescently labeled animal model of Alzheimer's disease. (A) shows the results of confirming that the animal model can label glutamatergic neurons red, and (B) shows the level of glutamatergic neuron activity induced by the tyrosine-glutamine (YQ) peptide diet. * indicates a statistically significant decrease in glutamatergic neuron activity in the glutamatergic neuron fluorescently labeled animal model of Alzheimer's disease (3xTG-vGluT2-Cre::tdTomato) compared to the control group (WT), p<0.05. # indicates a statistically significant increase in glutamatergic neuron activity in the peptide diet group (YQ) compared to the normal diet group (ND), p<0.05. [Figure 16] Figure 1 shows the seizure levels (A, B) and the areas under the curves (C, D) of A and B induced by treatment with tyrosine-glutamine (YQ) or tyrosine-tryptophan (YW) peptides in an animal model of epileptic seizures. *, **, and *** indicate a statistically significant reduction in seizure levels in the peptide-treated groups (5xL-Tyr, 3xYQ, YQi.p, 1xYW, or 3xYQ) compared with the normal diet group (ND), where *, P<0.05, **, and *** indicate P<0.01 and P<0.001, respectively. [Figure 17] This figure shows the ROS / RNS levels in an animal model of epileptic seizures following treatment with tyrosine-glutamine (YQ) or tyrosine-tryptophan (YW) peptides. * indicates a statistically significant increase in ROS / RNS levels in the kainic acid-treated group compared with the control group (CTL), p<0.05; # indicates a statistically significant decrease in ROS / RNS levels in the peptide diet groups (3xYW or 3xYQ) in the kainic acid-treated group compared with the normal diet group (ND), p<0.05. [Figure 18]This figure shows the activity of glutamine synthetase in animals treated with tyrosine-glutamine (YQ) or tyrosine-tryptophan (YW) peptides in an epileptic seizure model. *** indicates a statistically significant decrease in glutamine synthetase activity in the kainic acid-treated group compared to the control group (CTL), p<0.001. # and ## indicate a statistically significant increase in glutamine synthetase activity in the peptide-treated groups (3xYQ, YQi.p, 1xYW, 3xYW, or 3xYQ) in the kainic acid-treated group compared to the normal diet group (ND), p<0.05, ##<0.01. [Figure 19] Figure 1 shows the effects of tyrosine-glutamine (YQ) peptide treatment on neuronal count (A), SOD activity (B), and catalase activity (C) in an ischemic stroke animal model. Sham indicates the sham-operated group, and vehicle indicates the 0.9% saline-injected group. * and *** indicate a statistically significant decrease in neuronal count, SOD activity, or catalase activity in the ischemic stroke animal model (ischemia) compared with the sham-operated group (sham). * indicates P<0.05, and *** indicates P<0.001. # indicates a statistically significant increase in neuronal count, SOD activity, or catalase activity in the peptide-treated group (YQ) compared with the 0.9% saline-injected group (vehicle) in the ischemic stroke animal model (p<0.05). [Figure 20] Immunohistochemical staining results were obtained to confirm neuronal death following treatment with tyrosine-glutamine (YQ) peptide in an animal model of ischemic stroke. Sham indicates the sham-operated group, vehicle indicates the 0.9% saline injection group, and ischemia indicates the animal model of ischemic stroke. NeuN stained pyramidal cells, Iba-1 stained astrocytes, and GFAP stained microglial cells. [Figure 21] The graph shows the results of body weight change (A) and food intake (B) over time following treatment with tyrosine-glutamine (YQ) peptide in a high-fat diet type 2 diabetes animal model. ND is the normal diet group, and HFD is the high-fat diet group. [Figure 22] These figures show the results of blood glucose levels measured by tyrosine-glutamine (YQ) peptide treatment over time in a high-fat diet-induced type 2 diabetes animal model. ND indicates the normal diet group, and HFD indicates the high-fat diet group. *** and **** indicate a statistically significant increase in blood glucose in the high-fat diet group (HFD) compared to the normal diet group (ND). *** indicates P<0.001, and **** indicates P<0.0001. ## and ### indicate a statistically significant decrease in blood glucose in the YQ peptide-supplemented high-fat diet group (HFD + 1xYQ or HFD + 3xYQ) compared to the high-fat diet group (HFD). ## indicates P<0.01, and ### indicates P<0.001. [Figure 23] This figure shows the results of glucose tolerance test (GTT) evaluation using a high-fat diet-induced type 2 diabetes animal model after tyrosine-glutamine (YQ) peptide treatment. (A) shows blood glucose levels over time, and (B) shows the area under the curve (AUC) in (A). *** indicates a statistically significant increase in glucose levels in the high-fat diet group (HFD) compared with the normal diet group (ND), with P<0.001. ## indicates a statistically significant decrease in glucose levels in the high-fat diet group (HFD+3xYQ) supplemented with YQ peptide compared with the high-fat diet group (HFD), with P<0.01. [Figure 24] These figures show the results of the insulin tolerance test (ITT) performed on a high-fat diet-induced type 2 diabetes animal model. (A) shows blood glucose levels over time, and (B) shows the area under the curve (AUC) in (A). *** indicates a statistically significant increase in glucose levels in the high-fat diet group (HFD) compared with the normal diet group (ND), p<0.001. #### indicates a statistically significant decrease in glucose levels in the high-fat diet group (HFD+3xYQ) supplemented with YQ peptide compared with the high-fat diet group (HFD), p<0.0001. [Figure 25]These figures show the effects of tyrosine-glutamine (YQ) peptide treatment on urine output (A) and fat mass (B) in a high-fat diet-induced type 2 diabetes animal model. * indicates a statistically significant increase in urine output or fat mass in the high-fat diet group (HFD) compared with the normal diet group (ND), with p<0.05. ## and #### indicate a statistically significant decrease in urine output or fat mass in the YQ peptide-supplemented high-fat diet group (HFD+3xYQ) compared with the high-fat diet group (HFD), with ## indicating P<0.01 and #### indicating P<0.0001. [Figure 26] Figure 1 shows the plasma insulin (A), ALT (B), and ROS / RNS (C) concentrations after treatment with tyrosine-glutamine (YQ) peptide in a high-fat diet-induced type 2 diabetes animal model. **, ***, and **** indicate statistically significant increases in plasma insulin, ALT, or ROS / RNS concentrations in the high-fat diet group (HFD) compared with the normal diet group (ND). **, ***, and **** indicate P<0.01, P<0.001, and P<0.0001, respectively. ## indicates statistically significant decreases in plasma insulin, ALT, or ROS / RNS concentrations in the YQ peptide-added high-fat diet group (HFD+3xYQ) compared with the high-fat diet group (HFD). ## indicates statistically significant decreases in plasma insulin, ALT, or ROS / RNS concentrations in the ... [Figure 27] This shows the results of H&E staining to confirm changes in liver tissue due to tyrosine-glutamine (YQ) peptide treatment in a high-fat diet type 2 diabetes animal model. ND is the normal diet group, and HFD is the high-fat diet group. [Figure 28] These results show that insulin receptor β (IRβ) expression levels were determined by treatment with tyrosine-glutamine (YQ) peptide in a high-fat diet-induced type 2 diabetes animal model. * indicates a statistically significant decrease in insulin receptor β (IRβ) expression levels in the high-fat diet group (HFD) compared to the normal diet group (ND), p<0.05. ## indicates a statistically significant increase in insulin receptor β (IRβ) expression levels in the high-fat diet group (HFD + 3xYQ) supplemented with YQ peptide compared to the high-fat diet group (HFD), p<0.01. [Figure 29]These figures show the effects of tyrosine-glutamine (YQ) peptide treatment on the urine albumin / creatine ratio (A), liver triglyceride content (B), and plasma triglyceride content (C) in a high-fat diet-induced type 2 diabetes animal model. * and ** indicate a statistically significant increase in the urine albumin / creatine ratio, liver triglyceride content, or plasma triglyceride content in the high-fat diet group (HFD) compared with the normal diet group (ND). * indicates P<0.05, ** indicates P<0.01. # and ## indicate a statistically significant decrease in the urine albumin / creatine ratio, liver triglyceride content, or plasma triglyceride content in the YQ peptide-supplemented high-fat diet groups (HFD + 1xYQ or HFD + 3xYQ) compared with the high-fat diet group (HFD). # indicates P<0.05, ## indicates P<0.01. [Figure 30] This figure shows the results of measuring plasma creatinine concentration (A) to evaluate kidney damage caused by tyrosine-glutamine (YQ) peptide treatment in an animal model of acute renal failure, and the expression levels of IL-1β (B), IL-6 (C), and MCP-1 (D) to confirm the inflammatory response in kidney tissue. "Sham" refers to the sham-operated group, "renal IR" refers to the acute renal failure-induced group, and "veh" refers to the water-administered group. ** indicates a statistically significant increase in creatinine concentration, IL-1β, IL-6, or MCP-1 expression in the water-administered acute renal failure-induced group (veh + renal IR) compared to the sham-operated group (sham), with p<0.01. # and ## indicate that the creatinine concentration, IL-1β, IL-6, or MCP-1 expression levels were statistically significantly reduced in the acute renal failure induced group administered YQ peptide (YQ+renal IR) compared to the acute renal failure induced group administered water (veh+renal IR), where # indicates P<0.05 and ## indicates P<0.01. [Figure 31]These results show the expression of nitrotyrosine and lipid peroxidation products (4-HNE) in kidney tissue following treatment with tyrosine-glutamine (YQ) peptide in an animal model of acute renal failure. * indicates a statistically significant increase in the expression of nitrotyrosine and lipid peroxidation products (4-HNE) in the water-treated acute renal failure-induced group (veh+renal IR) compared with the sham-operated group (sham), p<0.05. # indicates a statistically significant decrease in the expression of nitrotyrosine and lipid peroxidation products (4-HNE) in the YQ peptide-treated acute renal failure-induced group (YQ+renal IR) compared with the water-treated acute renal failure-induced group (veh+renal IR), p<0.05. [Figure 32] These figures show the results of investigating blood ammonia levels (A) and nitrotyrosine expression levels in liver tissue following treatment with tyrosine-glutamine (YQ) peptide in a hyperammonemia animal model. Control is the normal control group, and AOM is the azoxymethane-induced hyperammonemia group. * and ** indicate statistically significant increases in blood ammonia levels and nitrotyrosine expression levels in liver tissue in the water-administered hyperammonemia group (veh+AOM) compared with the normal control group (Control). * indicates P<0.05, and ** indicates P<0.01. # indicates statistically significant decreases in blood ammonia levels and nitrotyrosine expression levels in liver tissue in the YQ peptide-administered hyperammonemia group (YQ200+AOM) compared with the water-administered hyperammonemia group (veh+AOM), p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0013] To achieve the object of the present invention, the present invention provides a functional health food composition for preventing or ameliorating diseases caused by protein nitration, which contains a peptide having a terminal tyrosine or a food-scientifically acceptable salt thereof as an active ingredient.

[0014] The peptide having tyrosine at its terminus has tyrosine at both termini or one terminus, and the amino acids linked to the tyrosine may be polar / hydrophobic (e.g., tyrosine, glutamine, threonine), non-polar / hydrophobic (e.g., tryptophan, valine), or charged (e.g., arginine). The number of linked amino acids may be, but is not limited to, 1 to 29.

[0015] In the functional health food composition for preventing or ameliorating diseases caused by protein nitration according to the present invention, the protein is preferably any one selected from the group consisting of glutamine synthetase, insulin receptor β subunit, manganese superoxide dismutase, heat shock protein 60, Cu / Zn superoxide dismutase, and catalase, but is not limited thereto.

[0016] In addition, the disease caused by protein nitration is preferably any one selected from the group consisting of depressive disorder, anxiety disorder, stroke, epilepsy, seizures, cognitive impairment, Alzheimer's disease, dementia, type 2 diabetes, diabetic nephropathy, sarcopenia, dyslipidemia, obesity, non-alcoholic fatty liver disease, acute kidney injury, hyperammonemia, and hepatic encephalopathy, but is not limited thereto.

[0017] The health functional food composition for preventing or improving diseases caused by the nitration of proteins can be prepared in the form of any one selected from pills, tablets, capsules, powders, powders, granules, candies, syrups, and beverages, or can be added as an ingredient to food, and can be appropriately prepared by a conventional method.

[0018] Examples of foods to which the active ingredient of the present invention can be added include any one of meats, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, energy drinks, alcoholic beverages and vitamin complexes, and include all of the health functional foods in the usual sense. The health functional foods may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (such as cheese and chocolate), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonation agents used in carbonated beverages. Fruit pulp may also be included for the production of natural fruit juices and vegetable beverages. These ingredients may be used independently or in combination.

[0019] The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame.

[0020] The present invention also provides a pharmaceutical composition for preventing or treating diseases caused by protein nitration, which comprises a peptide having a tyrosine terminal or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] In the pharmaceutical composition for preventing or treating diseases caused by protein nitration of the present invention, the protein and the disease caused by protein nitration are as described above.

[0022] In addition to the peptide having a tyrosine terminal, the composition may further contain a pharmaceutically acceptable carrier, excipient, or diluent. The pharmaceutical composition of the present invention may be administered orally or parenterally, and for parenteral administration, it is preferable to select, but is not limited to, topical application to the skin or intraperitoneal, rectal, intravenous, intramuscular, or subcutaneous injection.

[0023] The pharmaceutical compositions of the present invention may be formulated using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid formulations are prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, are also used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspension solvents that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Suppository bases that can be used include witepsol, macrogol, twine 61, cacao butter, laurin butter, glycerol, gelatin, etc.

[0024] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0025] The compositions of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, either sequentially or simultaneously with conventional therapeutic agents, and may be administered as single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that provides the maximum effect at the minimum dose without side effects, and this can be easily determined by one skilled in the art.

[0026] The dosage of the composition of the present invention may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0027] The present invention also provides a composition for inhibiting the nitration of tyrosine in a protein, which comprises, as an active ingredient, a peptide terminated in tyrosine or a pharmaceutically acceptable salt thereof.

[0028] In the composition for inhibiting tyrosine nitration in a protein of the present invention, the protein is as described above.

[0029] The present invention also provides a method for treating a tyrosine-terminated peptide with a protein containing a nitrated tyrosine to remove the nitro group from the nitrated tyrosine.

[0030] The present invention also provides a functional health food composition for preventing or ameliorating diseases caused by an increase in reactive oxygen species / reactive nitrogen species, which contains a peptide terminated in tyrosine or a food-scientifically acceptable salt thereof as an active ingredient.

[0031] The present invention also provides a pharmaceutical composition for preventing or treating diseases caused by an increase in reactive oxygen species / reactive nitrogen species, comprising a peptide terminated with tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient.

[0032] In the pharmaceutical composition for preventing or treating a disease caused by an increase in reactive oxygen species / reactive nitrogen species of the present invention, the disease caused by an increase in reactive oxygen species / reactive nitrogen species is preferably, but is not limited to, any one selected from the group consisting of depressive disorder, anxiety disorder, stroke, epilepsy, seizures, cognitive impairment, Alzheimer's disease, dementia, type 2 diabetes, diabetic nephropathy, sarcopenia, dyslipidemia, obesity, non-alcoholic fatty liver disease, acute kidney injury, hyperammonemia, and hepatic encephalopathy.

[0033] The present invention will be described in more detail below using examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail and that the scope of the present invention is not limited by them.

[0034] Materials and Methods 1. Peptide Synthesis To analyze the effects of the peptides according to the present invention, the following amino acids were used: glutamine (Q); L -Tyrosine ( L -Y) or D -Tyrosine ( D-Y), and peptides with tyrosine at the terminus include tyrosine-glutamine (YQ), glutamine-tyrosine (QY), tyrosine-tryptophan (YW), tryptophan-tyrosine (WY), tyrosine-threonine (YT), threonine-tyrosine (TY), tyrosine-arginine (YR), arginine-tyrosine (RY), tyrosine-valine (YV), valine-tyrosine (VY), tyrosine-tyrosine-glutamine (YYQ), glutamine-tyrosine-tyrosine (QYY), tyrosine-threonine-glutamine (YTQ), Glutamine-Threonine-Tyrosine (QTY), Tyrosine-Tryptophan-Glutamine (YWQ), Glutamine-Tryptophan-Tyrosine (QWY), Tyrosine-Glutamine-Glutamine (YQQ), Glutamine-Glutamine-Tyrosine (QQY), Tyrosine-Tyrosine-Tyrosine-Glutamine (YYYQ), Glutamine-Tyrosine-Tyrosine-Tyrosine (QYYY), Tyrosine-Tryptophan-Threonine-Glutamine (YWTQ), Glutamine-Tryptophan-Threonine-Tyrosine (QWTY), Tyrosine-Random 8 Amino Acids-Tyrosine (Y(A)8Y), Tyrosine-Random 18 Amino Acids-Tyrosine (Y(A) 18 Y) or tyrosine-28 random amino acids-tyrosine (Y(A) 28 The peptides used in the in vitro experiments were synthesized by peptron (purity ≥ 98%), and those used in the in vivo experiments were synthesized by GL Biochem (purity ≥ 95%).

[0035] 2. Animal Experiments In this study, 7-week-old C57BL / 6 male mice, 3xTG-AD mice (an Alzheimer's disease model), 4-week-old ICR male mice, or Mongolian gerbils (65-75 g) were housed at a temperature of 22-24°C, humidity of 50-70%, and a 12-hour light-dark cycle, with free access to diet and water.

[0036] The animals used in this study were maintained in accordance with the guidelines of the National Institutes of Health (NIH, Bechesda, MD, USA) and were operated under a protocol (GNU-161128-M0068) approved by the Gyeongsang National University Animal Care and Use Committee (GNU IACUC).

[0037] 3. Mouse Brain and Liver Tissue Sample Preparation Prefrontal cortex (PFC) and liver tissues were collected from mice anesthetized with CO2 gas, weighed, and then homogenized using a tissue homogenizer. The tissues were then centrifuged at 12,000 rpm at 4°C for 20 minutes, and the supernatant was removed to obtain PFC and liver tissue lysates.

[0038] 4. Preparation of diet supplemented with tyrosine-containing peptides To conduct animal experiments using the peptide diet of the present invention, mouse diets supplemented with tyrosine-containing peptides were prepared. The tyrosine-containing peptides used were tyrosine-glutamine (YQ), glutamine-tyrosine (QY), or tyrosine-tryptophan (YW), with molecular weights of 309.32 g / mol and 367.40 g / mol, respectively, and were added to a general diet (AIN-93G) (Table 1). [Table 1]

[0039] 5. Cognitive function test To analyze the long-term memory ability of animals fed the peptide diet of the present invention, an object recognition test (ORT) and an object location recognition test (OLT) were conducted (Figure 1). The test consisted of three stages: habituation, familiarization, and testing. Each animal was allowed to adapt to the test box for 10 minutes per day for two days. During the familiarization stage on the third day, the animals were asked to explore two identical objects for 10 minutes. Then, during the test stage on the fourth day, one of the two objects was replaced with a new object, and the animals were asked to explore for 10 minutes. The initial 5-minute recording was used as the data. The object recognition function (ORT) index was calculated using the following formula: Discrimination index = (NF) / (N+F) N: New thing search time F: Familiar object exploration time

[0040] 24 hours after completing the ORT, the location of the familiar object was changed and the subjects were asked to explore it for 5 minutes. The percentage of time spent exploring the relocated object out of the total object exploration time was calculated to calculate the object location recognition function (OLT) index.

[0041] Example 1: Analysis of the inhibitory effect on protein nitration 1-1 Analysis of the inhibitory effect on protein nitration in mouse brain and liver tissues Mouse brain and liver tissue lysates were used to analyze the inhibitory effect of the peptides of the present invention on protein nitration. Peroxynitrite (PN), which induces protein nitration, and the peptides of the present invention were added at a concentration of 2 mM to the tissue lysates. The mixture was vortexed for 5 seconds and incubated on ice for 10 minutes. Western blotting was then performed using anti-nitrotyrosine antibody (1:1,000), anti-insulin receptor β antibody (1:1,000), or anti-phosphorylated insulin receptor β antibody (1:1,000).

[0042] We confirmed that PN increased the number of proteins with increased tyrosine nitration in PFC and liver tissue. Western blot analysis of many bands confirmed that nitration of many proteins, except glutamine synthetase (GS) and insulin receptor β subunit (IRb), was increased. Furthermore, we confirmed that the nitration of proteins with increased tyrosine nitration by PN was reduced by treatment with a peptide containing a tyrosine (Y) at the terminus (Figure 2). Furthermore, in liver tissue, there was no difference in the expression of IRb protein between PN and the peptide containing a tyrosine (Y). However, phosphorylated-IRb protein expression was reduced by PN nitration and increased by treatment with a peptide containing a tyrosine (Y) at the terminus (Figure 3).

[0043] 1-2 Analysis of the nitration inhibitory effect of Cu / ZnSOD and MnSOD To analyze the inhibitory effect of the peptides of the present invention on the nitration of Cu / Zn superoxide dismutase (SOD-1) and manganese superoxide dismutase (SOD-2), an SOD colorimetric activity kit (Thermo Scientific, #EIASODC) was used. Human recombinant Cu / ZnSOD or MnSOD within the standard range was placed in a PCR tube, and the peptides of the present invention were added at a concentration of 1 mM. 1 mM peroxynitrite (PN) was added, mixed, and incubated on ice for 10 minutes. 25 μl of xanthine oxidase provided by the kit was then added, and the mixture was incubated at room temperature for 20 minutes. Absorbance was measured at 450 nm. The activity of Cu / ZnSOD or MnSOD was measured using the data obtained by subtracting the absorbance from the primary measurement from the secondary measurement.

[0044] As a result, we confirmed that the activity of Cu / ZnSOD or MnSOD was rapidly reduced by PN treatment, but that the activity of Cu / ZnSOD or MnSOD was restored by the peptide with a tyrosine terminal group (Figure 4).

[0045] 1-3 Analysis of the inhibitory effect of glutamine synthetase on nitration Mouse brain tissue lysate was used to analyze the inhibitory effect of the peptides of the present invention on glutamine synthetase nitration. The PFC tissue lysate was placed in a PCR tube, and the peptides of the present invention were added at a concentration of 1 mM. 1 mM peroxynitrite (PN) was added, mixed, and incubated on ice for 10 minutes. 50 μl of the sample was then placed in a 96-well plate and 50 μl of glutamine synthetase assay buffer (50 mM imidazole-HCl, pH 6.8, 25 mM L-glutamine, 12.5 mM hydroxylamine, 12.5 mM sodium arsenate, 1 mM MnCl2, 0.08 mM ADP) was added. The mixture was incubated at 37°C for approximately 40 minutes, and the absorbance was measured at 560 nm. The activity of glutamine synthetase was calculated from a standard curve prepared using γ-glutamylhydroxamate.

[0046] As a result, it was confirmed that the activity of glutamine synthetase was suddenly decreased by PN treatment, but that the activity of glutamine synthetase was restored by the peptide with tyrosine at the terminus. In particular, we confirmed that a long-chain peptide consisting of 30 amino acids also had the effect of restoring the activity of glutamine synthetase, whose activity had been reduced by nitration due to PN treatment (Figure 5).

[0047] 1-4 Analysis of the inhibitory effect of catalase on nitration To analyze the inhibitory effect of the peptides of the present invention on catalase nitration, Western blot analysis was performed using human recombinant catalase. 0.2 μg of human recombinant catalase and 2 mM of the peptides of the present invention were added to a PCR tube, and 1 mM peroxynitrite (PN) was added. The mixture was then mixed and incubated on ice for 10 minutes. SDS sample buffer was then added, the mixture was boiled for 5 minutes, and the gel was electrophoresed on an SDS-PAGE gel. The gel was then transferred to a PVDF membrane and Western blot analysis was performed using an anti-nitrotyrosine antibody (1:1000). As a result, we confirmed that the expression of nitrated catalase increased with PN treatment, whereas the expression of nitrated catalase decreased with the peptide containing tyrosine at the terminus (Fig. 6).

[0048] 1-5 Analysis of the inhibitory effect of nitration by measuring the refolding activity of HSP60 (heat shock protein 60) The activity of HSP60, a type of chaperone protein, is known to be inhibited by protein nitration, and various diseases related to this are known. Therefore, to analyze the inhibitory effect of the peptide of the present invention on HSP60 nitration, the HSP60 / HSP10 Glow-Fold Protein Refolding Kit (R&D Systems, #K-300) was used. The HSP60 solution provided by the kit was placed in a PCR tube, and the peptide of the present invention was added at a concentration of 1 mM. Then, 1 mM peroxynitrite (PN) was added, mixed, and incubated on ice for 10 minutes. The HSP10 solution provided by the kit, Mg 2+The ATP solution and Glow-Fold substrate protein were added and incubated at room temperature for 15-30 minutes. The incubated samples were then heated to 45°C for 7 minutes and immediately placed on ice. 50 μl of luciferin solution and 4 μl of sample were mixed in a 96-well plate, and luminescence was measured using a microplate reader within 1-2 minutes (Time 0). The remaining samples were incubated at 30°C for 60 minutes, after which 50 μl of luciferin solution and 4 μl of sample were mixed in a 96-well plate, and luminescence was measured using a microplate reader within 1-2 minutes (Time 60). The HSP60 refolding activity of each sample was analyzed using the difference in absorbance (RLU, relative light unit) measured at Time 0 and Time 60.

[0049] As a result, we confirmed that the refolding activity of HSP60 was rapidly decreased by PN treatment, but that it was restored by peptides with tyrosine at the terminus (Figure 7). Based on these results, we concluded that peptides with tyrosine at the terminus could be used to prevent or ameliorate diseases induced by decreased HSP60 activity.

[0050] Example 2: Analysis of GS nitration inhibitory effect and antidepressant effect in a chronic physical restraint stress animal model Twenty-eight 7-week-old C57BL / 6 male mice were divided into two groups (normal group and stress group). The stress group was individually forced into a restrainer for 2 hours (2:00–4:00 PM) every day for 15 days, resulting in chronic physical restraint stress. The normal (CTL) and stress (STR) groups were then re-classified into a nutritionally balanced general diet group (ND) and a diet supplemented with YQ or QY peptide (330 mg / kg) (PD). After completing the 15-day chronic physical restraint stress period, mice underwent a sucrose preference test (SPT), and blood and PFC tissue samples were collected for subsequent experiments.

[0051] (1) Analysis of GS nitration inhibitory effect GS activity and expression levels were measured using PFC homogenates from mice after chronic physical restraint stress as described above.

[0052] As a result, we confirmed that GS expression levels were similar, but that stress reduced GS activity, and that GS activity in the stressed groups was statistically significantly increased in the YQ or QY peptide diet group compared to the general diet group (Figures 8A, 8B, 9A, and 9B).

[0053] We also measured the level of tyrosine nitration in GS by immunoprecipitation (IP)-WB. Tyr-nitration immunoprecipitation analysis of GS was performed using anti-nitrotyrosine antibody (ab61392, Abcam) and Protein A / G Plus Agarose (Santa Cruz) according to the manufacturer's protocol.

[0054] As a result, we confirmed that stress resulted in a statistically significant increase in the level of tyrosine nitration in GS, and that in the stressed groups, the level of tyrosine nitration in GS in the YQ or QY peptide diet group was statistically significantly decreased compared to the general diet group (Figures 8C, 8D, 9C, and 9D).

[0055] (2) Antidepressant effect analysis After chronic physical restraint stress, blood and PFC tissue were collected from mice and analyzed for antidepressant effects. Blood was collected from the mice and centrifuged at 1,000 × g for 10 minutes at 4°C. The supernatant was removed and plasma was separated and diluted 1 / 20 with PBS. Plasma corticosterone concentrations were measured using a corticosterone EIA kit (Cayman) according to the manufacturer's method.

[0056] As a result, we confirmed that stress caused a statistically significant increase in plasma corticosterone levels, and that plasma corticosterone levels were reduced in the stressed groups in the YQ or QY peptide diet group compared to the general diet group (Figs. 10A and 11A).

[0057] Plasma diluted 1 / 3 with PBS and 50 μg of PFC tissue homogenate were also prepared. ROS / RNS (reactive oxygen species / reactive nitrogen species) concentrations in the plasma and PFC tissue were measured using a ROS / RNS assay kit (Cell Biolabs) according to the manufacturer's protocol.

[0058] As a result, we confirmed that stress caused a statistically significant increase in ROS / RNS concentrations in plasma and PFC tissue, and that in the stressed group, the ROS / RNS concentrations in plasma and PFC tissue were reduced in the YQ or QY peptide diet group compared to the general diet group (Figures 10C, 10D, 11C, and 11D).

[0059] Depression was also assessed by measuring sucrose preference in mice after 15 days of chronic physical restraint stress. The sucrose preference test was conducted for a total of 4 days. The same amount of 0.1 M sucrose and water was provided in a drinking trough of the same size and shape. On day 1, 0.1 M sucrose and water were provided for 24 hours. On day 2, the positions of the sucrose and water were swapped and provided for 24 hours. On day 3, 0.1 M sucrose and water were not provided. On day 4, the same amount of 0.1 M sucrose and water was again provided for 3 hours, then swapped and provided for another 3 hours. The amounts of sucrose and water consumed for 6 hours on day 4 were measured, and sucrose preference (%) was calculated using the following formula:

[0060] Sucrose preference (%) = sucrose intake / (water intake + sucrose intake) × 100 As a result, it was confirmed that stress reduced sucrose preference, and that in the stressed groups, the sucrose preference of the YQ or QY peptide diet group increased compared to the general diet group (FIGS. 10B and 11B).

[0061] Example 3 Analysis of cognitive function improvement in a chronic physical restraint stress animal model Twenty-eight 7-week-old C57BL / 6 male mice were divided into two groups (normal group and stress group) and fed a diet supplemented with tyrosine-containing peptides (1xYQ, 2xYQ, or 1xYW) for one week. The stress group was individually forced into a restrainer for two hours (2:00–4:00 PM) every day for two weeks, and subjected to chronic physical restraint stress. Subsequently, an object recognition test (ORT) was performed.

[0062] As a result, it was confirmed that stress reduced object recognition function, and that object recognition function increased in the stressed group in the YQ or YW peptide diet group compared to the general diet group (Figure 12).

[0063] Example 4 Analysis of cognitive function improvement in an animal model of Alzheimer's disease 4-1 Genetically engineered mouse model of Alzheimer's disease After examining the cognitive function of 2-month-old 3xTG-AD mice, a genetically engineered mouse model of Alzheimer's disease, and 62-month-old normal C57BL / 6 mice, the mice were divided into two groups and fed either normal diet (ND) or diet supplemented with a tyrosine-containing peptide (1xYQ). The cognitive function of 3xTG-AD mice was also examined at 8 months of age, the age at which mild cognitive impairment is known to appear, to examine the protective efficacy of the tyrosine peptide.

[0064] As a result, it was confirmed that the cognitive function of the dementia animal model was reduced compared to normal mice, and that the decline in cognitive function at 8 months of age was greater than that at 2 months of age. Furthermore, while there was no difference in the normal mouse group due to the peptide diet, in the dementia animal model, it was confirmed that the cognitive function of the dementia animal model was slightly increased by the YQ peptide diet (Figure 13).

[0065] In addition, hippocampal brain tissue was added to 100 μl of RIPA buffer solution (containing protease / phosphorylase inhibitors) per 10 mg of tissue mass and crushed for 1 minute using a glass bead blender. The supernatant was separated by centrifugation at 12,000 × g for 15 minutes at 4°C, diluted 10-fold with PBS, and used for reactive oxygen species / reactive nitrogen species (ROS / RNS) measurements. ROS / RNS concentrations were measured using the OxiSelect ROS / RNS Assay Kit (Cell Biolabs) according to the recommended experimental method.

[0066] As a result, it was confirmed that the ROS / RNS concentration in the hippocampal tissue of the dementia animal model was increased compared to the normal group, but was reduced by the YQ peptide (FIG. 14).

[0067] 4-2 Fluorescently labeled glutamatergic neurons in Alzheimer's disease model We crossed vGluT2-IRES-Cre::tdTomato mice, which have fluorescently labeled glutamatergic neurons, with 3xTG-AD mice, a genetically engineered mouse model of Alzheimer's disease, to construct 3xTG-vGluT2-Cre::tdTomato, a dementia model with fluorescently labeled glutamatergic neurons. We confirmed that this animal model can label glutamatergic neurons in red (Figure 15A).

[0068] 3xTG-vGluT2-Cre::tdTomato mice were fed a diet supplemented with a tyrosine-containing peptide (1xYQ) at 2 months of age, and spontaneous excitatory postsynaptic currents (sEPSCs) were measured at 6–8 months of age to assess glutamatergic neuronal activity. To record membrane currents, 200 μm-thick transverse brain slices were placed in a recording chamber infused with artificial cerebrospinal fluid (CSF) at 1.5–2 mL / min, and whole-cell voltage-clamp recordings were obtained from visualized glutamatergic neurons in the medial prefrontal cortex (mPFC) at a holding potential of -70 mV. Glutamatergic currents were isolated by the addition of picrotoxin (100 μM). All recordings were made at 30±2°C and the pipette solution consisted of 130 mM KCl, 5 mM CaCl2, 10 mM EGTA, 10 mM HEPES, 2 mM MgATP, 0.5 mM Na2GTP and 5 mM phosphocreatine.

[0069] As a result, it was confirmed that the activity of glutamatergic neurons in the dementia animal model was decreased compared to that in the normal group, but was increased by YQ peptide (Fig. 15B).

[0070] Example 5: Analysis of the inhibitory effect on excitotoxicity and oxidative stress in an animal model of epileptic seizures Four-week-old ICR male mice were fed a normal diet, a diet supplemented with tyrosine (L-Tyr), or a tyrosine-containing peptide (3xYQ, 1xYW, or 3xYW) for one week, and then administered 200 mg / kg YQ intraperitoneally (YQ i.p.). Kainic acid (KA) was dissolved in saline while heating in a water bath to prepare a 4.5 mg / ml kainic acid solution. The kainic acid solution was then intraperitoneally injected (36 mg / kg). Seizure levels and symptoms were recorded for two hours. Brain tissue (prefrontal cortex and hippocampus) was removed from surviving mice 16 hours or 5 days after kainic acid injection.

[0071] 5-1 Determining the level of seizures The seizure level was determined and recorded using the criteria in Table 2 below. [Table 2]

[0072] As a result, it was confirmed that the seizure level was significantly reduced in the peptide diet group compared to the normal diet group (FIG. 16).

[0073] 5-2 Reactive oxygen species / reactive nitrogen species (ROS / RNS) measurement Brain tissue samples from the hippocampus were crushed for 1 minute in 100 μl of RIPA buffer solution (containing protease / phosphorylase inhibitors) per 10 mg of tissue mass using a glass bead blender. The supernatant was separated by centrifugation at 12,000 × g for 15 minutes at 4°C, diluted 10-fold in PBS, and used for ROS / RNS assays. ROS / RNS concentrations were measured using the OxiSelect ROS / RNS Assay Kit (Cell Biolabs) according to the recommended experimental method.

[0074] As a result, it was confirmed that the ROS / RNS concentration in the hippocampal tissue of mice that survived the kainic acid injection was increased by kainic acid, but was reduced by the 3xYQ or 3xYW peptide (FIG. 17).

[0075] 5-3 Glutamine synthetase (GS) activity measurement Two microliters of hippocampal lysate was added to a 96-well plate, followed by 50 μl of 50 mM imidazole-HCl buffer (pH 6.8). 50 μl of GS activity assay buffer (50 mM imidazole-HCl, pH 6.8, 25 mM L-glutamine, 12.5 mM hydroxylamine, 12.5 mM sodium arsenate, 1 mM MnCl2, and 0.08 mM ADP) was added and incubated at 37°C for approximately 40 minutes. After the incubation, the standard substance γ-glutamylhydroxamate was added to empty wells to concentrations ranging from 0.391 to 25.0 mM. 100 μl of reaction stop solution (90 mM FeCl3, 1.8 N HCl, and 1.45% (w / v) trichloroacetic acid) was added to the samples and standards, and the absorbance was measured at a wavelength of 560 nm. The GS activity of each sample was determined by comparison with a standard curve. The GS activity was expressed as the amount of γ-glutamylhydroxamic acid produced as the final product, and its unit was μM / min / ug protein.

[0076] As a result, it was confirmed that the activity of glutamine synthetase in the hippocampal tissue of mice that survived the kainic acid injection was decreased by kainic acid, but increased by 3xYQ, 1xYW, 3xYW, or 3xYQ peptides (Figure 18).

[0077] Example 6 Analysis of the effects of suppressing neuronal death and promoting neuronal cell reproduction in an animal model of ischemic stroke Three-month-old male Mongolian gerbils were anesthetized with 3% isoflurane and maintained under 2.5% isoflurane gas. After disinfecting the neck, an incision was made to expose both common carotid arteries. A microvascular clip was used to occlude blood flow for 5 minutes, inducing ischemia and inducing stroke. Body temperature was maintained at 37.0 ± 0.5°C during application of the vascular clip. Sham-operated animals served as controls.

[0078] 6-1 Analysis of the effect of suppressing neuronal death After inducing ischemia, YQ peptide was intraperitoneally administered at a dose of 200 mg / kg three times every 24 hours during reperfusion. On the fourth day, the animals were sacrificed, and the brain tissue was extracted to measure the number of dead neurons.

[0079] As a result, it was confirmed that the number of neurons in the ischemic stroke animal model was decreased compared to the sham-operated group, but was increased by the YQ peptide (FIG. 19A).

[0080] 6-2 SOD and CAT activity measurement After inducing ischemia, YQ peptide was administered intraperitoneally at a dose of 200 mg / kg once during reperfusion. After 2 or 24 hours, the rats were anesthetized with avertin, the cranial cavity was opened, the brain was quickly removed, and the hippocampal tissue was excised, rapidly cooled in liquid nitrogen, and stored in a deep freezer. SOD and CAT activities were then measured using an SOD colorimetric activity kit and a catalase colorimetric activity kit.

[0081] As a result, it was confirmed that 24 hours after reperfusion in the ischemic stroke animal model, SOD and CAT activities were decreased compared to the sham-operated group, but increased by the YQ peptide (FIGS. 19B and 19C).

[0082] 6-3 Immunohistochemical staining to confirm neuronal cell death Immunohistochemical staining was performed to confirm changes in neurons and glue cells and related factors in the hippocampus of an animal model of ischemic brain injury. After ischemia was induced, YQ peptide was administered intraperitoneally at 200 mg / kg three times every 24 hours during reperfusion. A control group was administered vehicle (0.9% saline) instead of the peptide. On day 4, the animals were sacrificed, and brain tissue was removed and fixed in 4% paraformaldehyde at 4°C. The tissue was then immersed in 10, 20, or 30% sucrose solution, frozen in liquid nitrogen, and serially sectioned at 30 μm thickness. The tissue was then placed in a cryopreservative solution and stored at -20°C. Brain tissue sections were washed three times for 10 minutes each with 0.01M PBS and then incubated with 0.3% H2O2 for 30 minutes to eliminate endogenous peroxidases present in the tissue. To prevent nonspecific immune reactions, sections were incubated with 5% normal serum corresponding to each antibody for 30 minutes. Then, sections were incubated overnight at room temperature with anti-pyramidal cell-associated antibody (NeuN), anti-astroglial cell-associated antibody (Iba-1), and anti-microglia cell-associated antibody (GFAP) at the appropriate dilutions. After incubation, the sections were incubated with a biotin-conjugated secondary antibody for 2 hours and then with ABC solution for 1 hour. The incubated tissues were developed using a 3,3'-DAB kit, smeared on glass slides, dried at room temperature for 12 hours, dehydrated, cleared, mounted in DPX mounting solution, and examined under a microscope (Olympus BX53).

[0083] As a result, we observed that almost no neurons were observed in the hippocampal CA1 region in the control group (0.9% sarin group), but that approximately 60.7% of cells survived in the YQ peptide group compared to the sham-operated group. Furthermore, in the ischemic stroke animal model, glial cells with expanded cytoplasm and extended cell processes were observed in the control group (0.9% sarin group), but in the YQ peptide group, the morphological changes in glial cells were not significant and they showed a morphology similar to that of the sham-operated group (Figure 20).

[0084] Based on these results, it was found that peptides with tyrosine at the end suppress oxidative damage and immune responses in brain tissues caused by ischemia, thereby preventing the death of neurons.

[0085] Example 7 Analysis of the effect of enhancing insulin sensitivity in a high-fat diet type 2 diabetes animal model b Three-week-old C57BL / 6 male mice were purchased from Coretec and maintained in an animal room at constant temperature (22 ± 2°C), humidity (50 ± 5%), and a 12-hour photoperiod. They were housed two mice per cage. After a one-week adaptation period, mice were divided into four groups and fed a normal diet (ND), a high-fat diet (HFD, 60% kcal fat), or a high-fat diet supplemented with YQ peptide (HFD + 1xYQ or HFD + 3xYQ) for 17 weeks.

[0086] 7-1 Body weight, food intake and fasting blood glucose measurement The body weight and food intake of each animal were measured every other week, and at 1, 5 or 8 weeks after the start of feeding, fasting blood glucose was measured using a blood glucose measuring device (Accu-Check) after a 12-hour fast.

[0087] As a result, body weights gradually increased in all experimental groups, and on the same day, the HFD group was found to have a higher body weight than the ND group, and the HFD+3xYQ group was found to have a lower body weight than the HFD group (Figure 21A).Food intake was found to be lower in the HFD, HFD+1xYQ, and HFD+3xYQ groups than in the ND group, and there was almost no difference between the HFD, HFD+1xYQ, and HFD+3xYQ groups (Figure 21B).

[0088] It was also confirmed that blood glucose levels increased in the HFD group compared to the ND group, and decreased over time in the HFD+1xYQ group or HFD+3xYQ group compared to the HFD group (Figure 22).

[0089] 7-2 Glucose tolerance test (GTT) and insulin sensitivity test (ITT) After the start of feeding, a GTT test was performed at 15 weeks and an ITT test at 16 weeks. For the GTT, after a 12-hour fast, 2 g / kg of glucose solution was injected intraperitoneally, and blood was collected from a vein at 0, 20, 60, 90, and 120 minutes to measure blood glucose. For the ITT, after a 6-hour fast, 0.75 U / kg of insulin was injected intraperitoneally, and blood was collected from a vein at 0, 15, 30, 60, and 120 minutes to measure blood glucose.

[0090] The results of the glucose tolerance and insulin sensitivity tests confirmed that the blood glucose level in the HFD group was increased compared to the ND group, and that the blood glucose level in the HFD+3xYQ group was decreased compared to the HFD group (Figs. 23 and 24).

[0091] 7-3 Measurement of urine volume and fat mass Urine volume was measured by measuring the amount of urine (ml) collected in metabolic cages for 16 hours, and fat mass was measured by EchoMRI. TM The weight was measured from each mouse using a machine and expressed as a percentage of body weight.

[0092] As a result, it was confirmed that the urine volume and fat mass were increased in the HFD group compared to the ND group, and that the urine volume and fat mass were decreased in the HFD+3xYQ group compared to the HFD group (FIG. 25).

[0093] 7-4 Plasma biochemical analysis After anesthesia, blood was collected from the descending aorta and centrifuged at 3,000 rpm for 15 minutes to separate plasma. Insulin levels were measured using an ELISA kit (Crystal Chem, Ultra Sensitive Mouse Insulin ELISA Kit), alanine aminotransferase (ALT) levels were measured using an assay kit (IVD Lab Co., ChemiLab GPT(ALT) assay kit), and ROS / RNS levels were measured using an assay kit (Cell Biolabs, Inc., OxiSelect TM Measurements were performed using the In Vitro ROS / RNS Assay Kit.

[0094] As a result, it was confirmed that plasma insulin, ALT, and ROS / RNS concentrations were increased in the HFD group compared to the ND group, and plasma insulin, ALT, and ROS / RNS concentrations were decreased in the HFD+3xYQ group compared to the HFD group (Figure 26).

[0095] Based on the above results, it was found that peptides with tyrosine at the end can be used to prevent or treat non-alcoholic liver disease and chronic metabolic diseases caused by reactive oxygen / nitrogen species.

[0096] 7-5 Histological analysis After anesthesia, liver tissue was removed and rapidly cooled in liquid nitrogen. Some sections were fixed in 10% formalin and then paraffin-blocked to prepare 5-μm-thick histological slides. The tissues were then stained with H&E (hematoxilyn and eosin) and imaged under a microscope (Olympus, CKX41).

[0097] As a result, traces of lipid droplets within hepatocytes and tissue fibrosis were observed in the livers of the HFD group, while the HFD+3xYQ group maintained a liver tissue morphology similar to that of the ND group (Figure 27).

[0098] 7-6 Measurement of insulin receptor expression level Loss of muscle insulin receptors in metabolic diseases is known to cause sarcopenia by reducing insulin signal sensitivity and limiting the muscle's ability to use glucose. Liver tissue was homogenized in RIPA buffer, and protein was quantified using the BCA method and Western blot analysis was performed using an anti-insulin receptor-β (IRβ) antibody.

[0099] As a result, it was confirmed that the expression level of insulin receptor β was decreased in the HFD group compared to the ND group, and that the expression level of insulin receptor β was increased in the HFD+3xYQ group compared to the HFD group (Figure 28).

[0100] Based on the above results, it was found that peptides with tyrosine at the end increase the expression level of insulin receptor β, thereby having a preventive effect on sarcopenia caused by metabolic diseases.

[0101] 7-7 Measurement of albumin / creatine content and triglyceride content It is known that impaired renal function leads to an increase in albumin content and a decrease in creatine content in urine. Therefore, the albumin and creatine content in the urine of each experimental animal was measured using a mouse albumin ELISA kit (Abcam) and a creatine assay kit (Abcam), and the albumin / creatine ratio was calculated. Furthermore, 10 μl of plasma or 40 mg of homogenized liver tissue was centrifuged at 10,000 g for 10 minutes, and the supernatant was separated and the amount of triglycerides in the plasma or liver was measured using a triglyceride (TG) measurement kit (Cayman).

[0102] As a result, it was confirmed that the albumin / creatine ratio and triglyceride amount were increased in the HFD group compared to the ND group, and that the albumin / creatine ratio and triglyceride amount were decreased in the HFD+1xYQ or HFD+3xYQ groups compared to the HFD group (Figure 29).

[0103] Based on the above results, it was found that peptides with tyrosine at the terminus suppress the decline in kidney function caused by diabetes and are effective in preventing diabetic nephropathy, one of the diabetic complications, and are effective in preventing or treating dyslipidemia, a phenomenon in which triglycerides in the blood increase abnormally.

[0104] Example 8: Analysis of the inhibitory effect of protein nitration in an animal model of acute renal failure induced by renal ischemia-reperfusion Male C57BL / 6 mice weighing 23–25 g were housed in a pathogen-free room maintained at constant temperature and humidity, with free access to water and food. They were divided into three groups: 1) a control group (sham), 2) a water-administered renal ischemia-reperfusion-induced group (veh + renal IR), and 3) a YQ peptide-administered renal ischemia-reperfusion-induced group (YQ + renal IR). YQ (100 mg / kg) was orally administered once daily for 4 days, and renal ischemia was induced 30 minutes after oral administration on the fourth day. Renal ischemia was induced by an abdominal incision and clamping of bilateral renal vascular pedicles using microvascular clamps (Müller atraumatic vascular clamps). After 25 minutes of ischemia, the clamps were removed and reperfusion was initiated. The control group (sham) underwent all surgical procedures identically, except for the induction of ischemia via clamping. After 24 hours of reperfusion, the experimental animals were sacrificed, blood was collected from the heart, and kidney tissues were excised.

[0105] 8-1 Blood creatinine concentration measurement The collected blood was centrifuged at 3,000 rpm for 15 minutes to separate the plasma, and the blood creatinine concentration, an indicator of kidney damage, was measured using the Jaffe method. The Jaffe method involves reacting the plasma sample with picric acid at 510 nm, measuring the absorbance, and then terminating the reaction with 60% acetic acid. The creatinine concentration was calculated as the difference between the absorbance and the absorbance, and expressed in mg / dL.

[0106] As a result, plasma creatinine was significantly increased 24 hours after renal ischemia and reperfusion compared to the control group (sham), and it was confirmed that such an increase was statistically significantly suppressed by administration of YQ peptide (Figure 30A).

[0107] 8-2 Real-time qPCR analysis Total RNA was extracted from kidney tissue using the Trizol method, and cDNA was synthesized using the RevertAid reverse transcription system (Thermofisher). Quantitative PCR (qPCR) for inflammatory cytokines (IL-1β, IL-6, and MCP-1) was performed using the CFX Connect Real-Time PCR System (Bio-Rad) with iQ SYBR Green Supermix (Bio-Rad). The relative amounts of target mRNA normalized to GAPDH were calculated using qPCR. -△Ct The primers used in the experiment are listed in Table 3 below. [Table 3]

[0108] As a result, the expression levels of IL-1β, IL-6, and MCP-1, which are indicators of the inflammatory response, which is the main pathogenic mechanism of acute renal failure, were significantly increased 24 hours after renal ischemia and reperfusion compared to the control group (sham), and it was confirmed that such increases were statistically significantly suppressed by administration of YQ peptide (Figure 30B, C, D).

[0109] 8-3 Measurement of expression levels of nitrated proteins and lipid peroxidation products in kidney tissue Kidney tissues were homogenized in RIPA buffer, and protein was quantified by the BCA method, followed by Western blotting using antibodies against nitrotyrosine and 4-hydroxynonenal (4-HNE), a product of lipid peroxidation. As a result, 24 hours after renal ischemia and reperfusion, the amount of tyrosine-nitrated proteins in kidney tissue increased compared to the control group (sham), and this increase was significantly reduced by administration of YQ peptide (Figure 31).

[0110] Example 9 Analysis of the effect of suppressing blood ammonia in a hyperammonemia animal model Thirteen-week-old male C57BL / 6 mice were housed in a pathogen-free room maintained at constant temperature and humidity, with free access to water and food. They were divided into three groups: (1) a control group, (2) a water-administered hyperammonemic group (veh+AOM), (3) a 100 mg / kg YQ peptide-administered hyperammonemic group (YQ100+AOM), and (4) a 200 mg / kg YQ peptide-administered hyperammonemic group (YQ200+AOM). YQ peptide was orally administered once daily for 4 days. On the fourth day, hyperammonemic control was induced by intraperitoneal injection of azoxymethane (AOM, 100 mg / kg) 2 hours after oral administration. 12 hours after AOM administration, 200 μl of 0.5% glucose in saline was injected intraperitoneally to prevent dehydration. Four hours later, the animals were sacrificed, blood was collected from the heart, and liver tissue was excised.

[0111] 9-1 Blood ammonia analysis Ammonia is a major metabolite of amino acids and nucleic acids. The key enzymes of the iodine cycle, which convert ammonia to iodine, are found only in hepatocytes. Liver tissue is also rich in GS and SOD, which reduce ammonia levels, and catalase, which removes reactive oxygen species. Therefore, changes in blood ammonia levels in the blood vessels that pass through the liver and into the brain are important indicators of hepatic encephalopathy. To measure blood ammonia levels, we used a PocketChem BA PA-4140 (Arkray, Japan), a single-wavelength reflectometry analyzer. After 20 μl of blood was incubated on a test strip at room temperature for 3 minutes, it was inserted into the analyzer and absorbance was measured at a wavelength of 635 nm (LED). Results were displayed in μg / dL.

[0112] As a result, the water-administered hyperammonemia group (veh+AOM) showed a 3.6-fold increase in blood ammonia concentration compared to the control group (Control), and it was confirmed that administration of YQ peptide statistically significantly suppressed the increase in blood ammonia caused by AOM (Figure 32A).

[0113] 9-2 Measurement of nitrated protein expression in liver tissue Liver tissue was homogenized in RIPA buffer, and protein was quantified by the BCA method, followed by Western blotting using an antibody against nitrotyrosine. As a result, nitrotyrosine protein increased in liver tissue after AOM administration compared to the control group, and this increase was significantly reduced by administration of YQ peptide. This result indicated that YQ peptide inhibits the nitration of proteins in the liver, enabling the removal of ammonia that has entered the liver (Figure 32).

[0114] [Statistical processing] All data herein are presented as mean ± standard deviation and were statistically analyzed by one-way analysis of variance (ANOVA) using Dunnett's Multiple Comparison Test or Student's t test using GraphPad Prism 5 (GraphPad Software).

Claims

1. A pharmaceutical composition for inhibiting nitration in a protein having a tyrosine moiety or removing a nitro group from a nitrated tyrosine, comprising a peptide having a terminal tyrosine or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the protein is any one selected from the group consisting of glutamine synthetase, insulin receptor β subunit, manganese superoxide dismutase, heat shock protein 60, Cu / Zn superoxide dismutase, and catalase.

2. The pharmaceutical composition according to claim 1, wherein the tyrosine-terminated peptide has tyrosines at both or one of its termini, and the number of amino acids linked to the tyrosines is 1 to 29.

3. The pharmaceutical composition according to claim 1, wherein the tyrosine-terminated peptide or a pharmaceutically acceptable salt thereof reduces reactive oxygen species / reactive nitrogen species.

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