KR101500335B1 - Therapeutic agents targeting p47phox or p67phox for stress disorder and method of screening thereof - Google Patents

Abstract

The present invention relates to a method for producing a stress-sensitive response modifier, a food or feed for improving stress, a method for producing the same, a pharmaceutical composition for preventing or treating stress disorder, a method for producing the same, A method for screening a stress-sensitive response modifier, and a method for providing information for diagnosing a stress disorder caused by a stress-sensitive reaction or chronic stress. According to the present invention, drugs using the mechanism of inhibiting the expression / activity / function of p47phox or p67phox can be developed. Drugs screened by the method of the present invention can be used for the treatment of stress, particularly depression , Degenerative brain diseases, and the like.

Description

TECHNICAL FIELD The present invention relates to a therapeutic agent for stress disorder targeting p47phox or p67phox and a screening method thereof.

The present invention relates to a method for producing a stress-sensitive response modifier, a food or feed for improving stress, a method for producing the same, a pharmaceutical composition for preventing or treating stress disorder, a method for producing the same, A method for screening a stress-sensitive response modifier, and a method for providing information for diagnosing a stress disorder caused by a stress-sensitive reaction or chronic stress.

Modern industrial society is changing rapidly and complexly structurally and functionally. In addition, there is an enormous amount of information coming out daily, which is difficult for an individual to digest. Most of the modern people who have to adapt to new technology and new environment feel a lot of physical and psychological burden. This stress is called stress.

When the human is subjected to physical and psychological stress by the stimulation of the internal and external environment, the human body needs to use brain corticotropin releasing (LC-NE: locus ceruleus norepinephrine) and corticotropin releasing hormone (CRH) hormone. LC-NE activates the sympathetic nervous system and suppresses the action of parasympathetic nervous system, which increases blood pressure, heart rate, respiration, and not only expands the bronchi, but also increases sweating that causes hair to creep and hairy. CRH secreted from the hypothalamus releases the adrenocorticotropic hormone (ACTH) from the anterior pituitary gland to the general systemic circulation. When this ACTH migrates along the blood and binds to the adrenal cortical ACTH receptor, it synthesizes a secondary signaling cAMP called cAMP. The cAMP-activated protein kinase PKA (protein kinase A) activates the glucocorticoid synthase protein located in mitochondria. The glucocorticoid synthase protein then uses cholesterol to produce stress hormones. Cortisol, corticosterone, cortisone, and the like are examples of typical glucocorticoids. The stress pathway to the CRH-ACTH-glucocorticoid is commonly referred to as the hypothalamus-pituitary gland-adrenal gland (HPA) axis. Glucocorticoids regulate carbohydrate metabolism, including gluconeogenesis, glycogen storage, and elevated blood glucose levels in the liver. In addition, glucocorticoids have anti-inflammatory effects by inhibiting the expression of various immune substance-producing genes of immune cells responsible for immunity. In addition, when the hormone content in the blood increases, negative feedback acts on the pituitary gland to suppress ACTH secretion.

While the body's acceptable levels of stress will act as an appropriate stimulant for our body, strong and persistent psychological and physical stresses beyond acceptable limits induce glucocorticoid overdose for prolonged periods of time. Glucocorticoids, which are secreted in such a long period of time, degrade the body's immune system, which in turn reduces the resistance to external infections and causes various diseases in our bodies. In addition to diminishing immunity, it also inhibits brain cell damage and regeneration, resulting in insomnia, lethargy, memory loss, depression, as well as suicidal impulses.

Depression, on the other hand, is classified as a type of mood disorder with bipolar disorder and has psychological and physical symptoms including stagnation mood, anorexia, sleep disturbances, nonsocial, fatigue, and impaired concentration (DSM-IV-TR) It is one of the most common forms of mental illness. Depression has a lifetime prevalence of 15%, especially in women, and is a serious disease that causes changes in emotions, thoughts, physical condition, and behavior. The World Health Organization (WHO) predicts by 2020 that depression will be the number one disease of all ages. In addition, depression often leads to suicide, and if the depression worsens to "the first disease of the future" in 2020, it can be said that mortality due to depression can be higher than the mortality caused by physical diseases like cancer. The pathophysiology was not revealed.

The major cause of depression is hormonal imbalance, which is the main cause of serotonin. Serotonin is a neuro-metabolite found in cerebrospinal fluid. If it is deficient, it causes anxiety, anxiety, and impulsivity. Therefore, antidepressants are associated with the action of serotonin.

Currently, antidepressants used in clinical practice are mostly drugs (SSRI, SNRI, SDRI, TCAs, MAOIs, etc.) that increase the pharmacological action of monoamine neurotransmitters at nerve synapses. Although these drugs clearly demonstrate antidepressant effects depending on the patient, paradoxically, their pharmacological mechanism is unclear in view of the fact that their efficacy is generally observed over a period of 2-3 weeks. In the case of the above-mentioned drugs, there is a report that the antidepressant effect can be reduced by reacting with an anti-inflammatory agent when administered together with an anti-inflammatory agent. In addition, the drugs may cause side effects such as vomiting, decreased sexual desire, insomnia, fatigue, obesity, and suicidal tendency. In the case of 30% of depressed patients, currently used drugs have no effective effect at all.

As described above, the existing therapeutic agents for treating depression by increasing the pharmacological action of the monoamine-based neurotransmitters have many side effects, and their therapeutic effects are limited. In addition, There is a desperate need for the development of a therapeutic system comprising a drug based on a drug.

Accordingly, the present inventors confirmed that expression of p47phox or p67phox gene present in nerve cells or blood is continuously increased in an animal model subjected to chronic stress or in neurons treated with stress hormone, compared with a control group without stress Based on this, it can be screened for a stress-sensitive response modifier targeting p47phox or p67phox. Moreover, by confirming that substances capable of inhibiting the expression, function, or activity of p47phox or p67phox can be used to prevent or treat stress disorder Thereby completing the present invention.

It is an object of the present invention to provide a method for producing a stress-sensitive reaction mitigating agent.

Another object of the present invention is to provide a food or feed for improving stress, and a method for producing the same.

Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating stress disorder, a method for producing the same, and a method for preventing or treating stress disorder.

It is still another object of the present invention to provide a method for screening a stress-sensitive response modifier.

It is another object of the present invention to provide a method for providing information for diagnosing a stress disorder caused by a stress sensitive response or chronic stress.

In order to solve the above problems, in one aspect, the present invention provides a method for producing a stress-sensitive reaction modifier comprising: (1) mass-producing a stress-sensitive reaction modifier; And (2) extracting a part of the produced stress-sensitive relaxation emollient to confirm whether or not it suppresses at least one of expression, activity and function of p47phox or p67phox. do.

The present invention also relates to a method for identifying a test substance, comprising: (1) confirming whether a test substance inhibits one or more of expression, activity, and function of p47phox or p67phox; And (2) mass-producing a test substance which has been confirmed to inhibit at least one of the expression, activity and function of p47phox or p67phox in the above step.

In another aspect, the invention provides a stress-improving food or feed containing a stress sensitive response modifier that inhibits one or more of the expression, activity, and function of p47phox or p67phox.

In another aspect, the present invention relates to a method for producing a stress-improving food or feed, which comprises the step of confirming whether or not a stress-improving food or feed inhibits at least one of expression, activity, and function of p47phox or p67phox ≪ / RTI >

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating stress disorder, which comprises, as an active ingredient, a substance that inhibits at least one of expression, activity, and function of p47phox or p67phox.

In another aspect, the present invention provides a method for treating or ameliorating a disorder or condition selected from the group consisting of a stress disorder characterized by comprising determining whether the pharmaceutical composition for the prevention or treatment of a stress disorder or its efficacy inhibits one or more of the expression, activity and function of p47phox or p67phox Or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method for preventing or treating a stress disorder by administering to a subject a substance that inhibits one or more of expression, activity, and function of p47phox or p67phox.

In another aspect, the present invention provides a method for the treatment of cancer, comprising: (1) providing a cell expressing p47phox or p67phox; (2) a second step of bringing the test substance into contact with the cell; And (3) a third step of screening candidate substances in which at least one of the expression, activity, and function of the p47phox or p67phox is inhibited in cells contacted with the test substance as compared with cells not in contact with the test substance A method for screening a stress sensitive response modifier.

In another aspect, the present invention provides a method for diagnosing a stress disorder caused by a stress-sensitive or chronic stress, which comprises measuring the expression, activity and function of p47phox or p67phox of a biological sample isolated from an individual, Provide a method of providing.

Hereinafter, the present invention will be described in detail.

(1) mass-producing a stress-sensitive reaction mitigating agent; And (2) extracting a part of the produced stress-sensitive reaction mitigating agent to confirm whether or not it suppresses at least one of expression, activity and function of p47phox or p67phox. will be.

The present invention also relates to a method for identifying a test substance, comprising: (1) confirming whether a test substance inhibits one or more of expression, activity, and function of p47phox or p67phox; And (2) mass-producing a test substance which has been confirmed to inhibit at least one of the expression, activity and function of p47phox or p67phox in said step.

The p47phox of the present invention is preferably a subunit of NADPH (Nicotinamide Adenine Dinucleotide Phosphate) oxidase, NCBI Accession No. mouse is NM_010876.2, and human is NM_000265.4

The p67phox of the present invention is preferably a subunit of NADPH oxidase, NCBI Accession No. mouse is NM_010877.4, and human is NM_001127651.2

The NADPH oxidase is a protein complex that transfers electrons through a biofilm. It transfers electrons from NADPH to oxygen molecules to generate superoxidized (Reactive Oxygen Species, ROS). The NADPH oxidase complex contains Rho guanosine triphosphatase (GTPase) (Rac1 or Rac2 (Rho-related C3 botulinum toxin substrate) and 5 gp91-phox (Nox2) including the "phox" , p40phox, p47phox, and p67phox. For activation, it is necessary to transfer the p40phox, p47phox and p67phox present in the cytoplasm to the membrane, in which phosphorylation of p47phox is essential. (Fig. 4B, C).

The term "stress-sensitive response" in the present invention means a reaction that can be caused by stress. For example, it is known that stress causes the locus ceruleus norepinephrine or corticosteroid hormone free hormone (CRH) : corticotropin releasing hormone (CNS) is a secreted or directly or indirectly generated reaction or stress, which is directly or indirectly caused by glucocorticoid hormone secreted by the hormone. In addition, the present invention provides a method for treating or preventing a disease caused by stress, such as depression, anxiety, fear memory, mental panic, obsessive compulsive disorder, posttraumatic stress disorder, schizophrenia, headache due to stress, neurodegenerative disease, Anorexia, gastritis, gastric ulcer, Alzheimer ' s dementia, Parkinson's disease, stroke, or stress-related reactions that occur prior to the onset of the disease.

The term "stress-sensitive reaction modifier" means a substance capable of reducing and alleviating the above-described "stress-sensitive reaction ", and may also be referred to as an anti-stress agent.

As used herein, the term " chronic stress "refers to bodily physiological responses and consequences resulting from sustained neurological and psychological brain damage when stress is repeatedly given over the medium to long term.

In the present invention, the chronic mechanism of repeated stress stress induces depression and anxiety, accumulation of a large amount of oxidative stress in the brain nerve, and a cause of severe mitochondrial dysfunction is the first to induce the molecular mechanism of activation of p47phox or p67phox by stress Respectively. In addition, we have shown that control of p47phox or p67phox can inhibit ROS overexpression and depression in the cranial nervous system.

In one embodiment of the present invention, it was confirmed that when chronic stress was applied to the mouse, lipid peroxidation and oxidative stress of the brain were accumulated over time (Example 3), and the expression of p47phox or p67phox was increased according to chronic stress (Examples 4 and 5), it was confirmed that a substance that inhibits the expression / function / activity of p47phox or p67phox was pretreated before stressing or depressive action due to chronic stress disappeared after treatment after stressing (Example 6 , 7). Based on this, the present invention can alleviate the stress-sensitive response by suppressing the expression, function, or activity of p47phox or p67phox at a stage before a stress reaction or a stress disorder induced by gradual accumulation of stress, The present invention also provides a method of inhibiting the expression, function, or activity of p47phox or p67phox, as well as preventing the occurrence of the disorder in advance.

The present invention relates to a method for confirming whether or not one or more of the expression, activity, and function of p47phox or p67phox is inhibited by extracting a part of a mass-produced stress-sensitive reaction mitigating agent after mass-producing a stress-sensitive reaction modifier, Stress-sensitive response modifiers can be prepared by performing steps to confirm that I have the ability to mitigate the stress sensitive response. Alternatively, the present invention can produce a stress-sensitive response modifier by selecting a test substance that is confirmed to inhibit at least one of the expression, activity, and function of p47phox or p67phox and mass-producing the same.

In the present invention, inhibiting at least one of the expression, activity, or function of p47phox or p67phox includes a substance that inhibits the expression / activity / function enhancement of p47phox or p67phox or the expression / activity / function of enhanced p47phox or p67phox .

In addition, in one embodiment of the present invention, it was confirmed that the expression of p47phox or p67phox was upregulated in the blood of an animal subjected to chronic stress. Based on this, it was confirmed that the expression, activity, and function of the p47phox or p67phox were suppressed Whether or not the step of ascertaining or inhibiting one or more of the expression, activity, function of p67phox of the test substance can be performed by measuring one or more of the expression, activity, or function of p47phox or p67phox in the blood of the animal.

In the present invention, the "test substance" is a substance that specifically inhibits the expression and / or activity of p47phox and / or p67phox or a combination thereof and includes low molecular weight compounds, high molecular weight compounds, nucleic acid molecules Aptamers), proteins, sugars, lipids, and the like. The test material comprises 10 or more bases of single or double stranded nucleic acid, e.g., 10, 11, 12, 14, 15 or more nucleic acid molecules such as siRNA, shRNA or miRNA. The test substance may also be a polypeptide having at least two amino acid residues, such as six, eight, ten, twelve, twenty or fewer, or more than twenty such as fifty (50) amino acid residues.

In the present invention, expression refers to transcription of a gene and expression of a transcript into a protein, and the term " activity " means a biological action that allows an expressed protein to function.

Whether or not the expression, activity, or function of p47phox or p67phox is inhibited by the present invention is not limited thereto, but it can be determined by measuring the expression level of p47phox or p67phox. The expression levels of p47phox or p67phox mRNA can be determined by RT-PCR, quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative real-time PCR, Northern blot and DNA or RNA chip, but the present invention is not limited thereto. In addition, the expression level of p47phox or p67phox protein can be measured using any method selected from the group consisting of Western blot, tissue immunostaining and tissue fluorescence staining, but the present invention is not limited thereto.

In another aspect, the present invention relates to a stress-improving food or feed containing a stress-sensitive response modifier exhibiting a property of suppressing at least one of the expression, activity, or function of p47phox or p67phox.

In another aspect, the present invention relates to a method for producing a stress-improving food or feed, which comprises the step of confirming whether or not a stress-improving food or feed inhibits at least one of expression, activity, and function of p47phox or p67phox ≪ / RTI >

In one example of the present invention, it was confirmed that ROS production was increased in chronic stressed mice and depressive behaviors appeared, and ROS production was increased in human neuroblastoma cell line treated with glucocorticoid, which is a stress hormone, And expression of p47phox or p67phox was increased in the cell line (Example 4). Also, as a result of injection of siRNA against p47phox, glucocorticoid-induced ROS production and peroxide production were decreased depending on the amount of treated siRNA (Example 6), and apoPOX or p67phox, Cyanine, an antidepressant, imipramine, was administered before and after chronic stress, and depression behavior and lipid peroxidation were reduced similarly to the non-stressed group (Example 7).

Based on the above experimental data, the present invention provides a stress-sensitive food or feed for improving stress, which comprises a stress-sensitive reaction modifier exhibiting a property of suppressing at least one of expression, activity, and function of p47phox or p67phox. The stress-improving food or feed can improve all symptoms caused by stress or symptoms that appear before a stress disorder induced by repetitive stress, and prevent stress disorder. The above-mentioned stress-sensitive response modifiers are not limited to compounds, natural products, novel proteins and the like, provided that they exhibit the property of suppressing at least one of expression, activity, and function of p47phox or p67phox. In particular, it also includes substances that inhibit the activity of p47phox or p67phox due to inability to migrate to the cell membrane.

In the present invention, the substance that inhibits at least one of the expression, activity, and function of p47phox or p67phox includes siRNA (small interfering RNA), shRNA (small interfering RNA) capable of specifically inhibiting transcription of p47phox and p67phox gene and / (small hairpin RNA) or miRNA (microRNA). Through RNA interference, for example, short-length interfering RNA (siRNA) binds to the transcript in a sequence-specific manner to form an RNA Induced Silencing Complex (RISC) Lt; RTI ID = 0.0 > silencing < / RTI > In one embodiment, the siRNA, shRNA or miRNA target the p47phox gene, which is one of the subunits of NADPH oxidase. The siRNA, shRNA or miRNA has a sequence that is highly complementary to its target sequence. A highly complementary sequence means at least about 70% complementarity, at least about 80% complementarity, at least about 90% complementarity, or about 100% complementarity with at least 15 consecutive bases in length of the target gene. In one embodiment, the p47phox siRNA or shRNA may include, but is not limited to, the siRNA used in p47phox silencing in the instant embodiments, the shRNA, Lenti-p47phox-shRNA, SiRNAs, shRNAs, and / or miRNAs that target various derived p47phox can be used as well as their biological equivalents, derivatives, and analogs. In one embodiment, the siRNA, shRNA and / or miRNA are derived from the human p47phox gene. In other embodiments, the siRNA, shRNA and / or miRNA may be between about 600 and 1200 bases, between about 700 and 1200 bases, between about 800 and 1200 bases, between about 900 and 1200 bases, based on the human-derived gene NM_000265.4 sequence. To 35 bases in length and can be designed using commercially available siRNA, shRNA or miRNA target design software.

Further, in another embodiment of the present invention, the stress-sensitive response modifier that inhibits one or more of the expression, activity, and function of the p47phox or p67phox is an antibody capable of recognizing p47phox, p67phox or a combination thereof. The term antibody as used herein includes complete antibodies, antigen-binding fragments of antibody molecules, and antibodies or fragments thereof functionally equivalent thereto. The antibodies of the invention may also be of the IgG, IgM, IgD, IgE, IgA or IgY type and may be of the IgGl, IgG2, IgG3, IgG4, IgA1 or IgA2 class or subclass thereof. Antibodies of the invention include monoclonal, polyclonal, chimeric, short chain, heterologous, humanized and humanized antibodies and active fragments thereof.

Further, in another embodiment of the present invention, the stress sensitive response modifier that inhibits one or more of the expression, activity and function of the p47phox or p67phox interferes with, inhibits, or inhibits the activity and / or action / function of p47phox, p67phox Lt; / RTI > The term polypeptide as used herein refers to a polymer of amino acids and does not refer to any particular length as long as it has this effect. Thus, for example, peptides, oligopeptides, proteins, and enzymes are also included in this scope. Polypeptides include those that have been modified by natural or artificial modifications such as glycosylation, acetylation, phosphorylation, and the like.

Also, stress-sensitive response modifiers that inhibit one or more of the expression, activity and function of p47phox or p67phox include aposinin, 4-hydroxy-3'-methoxy-acetophenone, N-vanillylnonamide (N- Vanillylnonanamide, Staurosporine, neopterin, diphenylene iodonium, or a combination thereof, but is not limited thereto.

The stress-sensitive reaction mitigating agent contained in the food or feed is preferably prepared by the above-described method for producing a stress-sensitive reaction mitigating agent.

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense. The health drink of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the food of the present invention. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. The proportion of such flesh is not critical but is generally selected in the range of 0.01 to 10 parts by weight per 100 parts by weight of the composition of the present invention. These components can be used independently or in combination.

According to the present invention, the feed can be classified into various kinds according to nutritive value, main ingredient, distribution, moisture content, processing type, etc., and the feed can be classified into various kinds such as a forage, a concentrated feed, a supplementary feed, a protein feed, a starch feed, Feed is available but not limited thereto, and includes feed additives. The feed may further contain a carrier such as poultry and livestock. In the present invention, the feed can be added as it is or a known carrier, stabilizer and the like can be added, and various nutrients such as vitamins, amino acids and minerals, antioxidants, antibiotics, antibacterial agents and other additives can be added , And the shape thereof may be a suitable state such as powder, granule, pellet, suspension and the like. When the feed of the present invention is supplied, it can be supplied to poultry and livestock singly or mixed with feed, and the feed can reduce the stress of poultry and domestic animals.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating stress disorder, which comprises, as an active ingredient, a substance which inhibits at least one of expression, activity and function of p47phox or p67phox.

In another aspect, the pharmaceutical composition for the prevention or treatment of stress disorder according to the present invention is characterized in that it is confirmed whether the pharmaceutical composition for preventing or treating stress disorder or an effective ingredient thereof inhibits one or more of the expression, activity and function of p47phox or p67phox And the like.

In another aspect, the present invention relates to a method for preventing or treating a stress disorder by administering to a subject a substance that inhibits one or more of expression, activity, and function of p47phox or p67phox.

In one embodiment of the present invention, it was confirmed that lipid peroxidation of the brain accumulates over time when sustained stress is applied to the mouse. Expression of p47phox or p67phox is increased according to chronic stress, and expression of siRNA against p47phox , It was confirmed that ROS production and peroxide production induced by glucocorticoids were reduced depending on the amount of siRNA treated (Example 6). In addition, treatment of apoptin, an antidepressant, imipramine, which suppresses the expression of p47phox or p67phox in stressed mice, resulted in a decrease in depressive behavior. In the p47phox mutant (+/-) mouse, + / +), It was also confirmed that depression was not caused by chronic stress (Example 7). Thus, a substance that inhibits one or more of the expression, activity, and function of p47phox or p67phox may prevent the disorder before the onset of the stress disorder and treat the stress disorder by enhancing the expression of PRX3 or TRX2 in the event of the onset of the stress disorder .

As the effective ingredient of the composition, there are no limitations on the types of compounds, natural products, novel proteins and the like, provided that they inhibit the expression, activity, or function of p47phox or p67phox. The substance that inhibits the expression, activity or function of p47phox or p67phox is preferably selected from the group consisting of aposinin, 4-hydroxy-3'-methoxy-acetophenone, N-vanillylononamide, But are not limited to, staurosporine, neopterin, diphenylene iodonium, or combinations thereof.

The term " prevention " in the present invention means all actions that inhibit or delay the symptoms of a stress-related disease by administration of the composition. In the present invention, the term " treatment " means any action that improves or alleviates symptoms of a stress-related disease by administering the composition.

The stress disorder refers to a disease that can be triggered or exacerbated by mental and / or physical chronic stress. Specifically, the stress disorder is caused by mental and / or physical chronic stress, depression, anxiety, fear memory, mental panic, A selected member selected from the group consisting of: a disorder selected from the group consisting of schizophrenia, schizophrenia, stress disorder, schizophrenia, headache due to stress, neurodegenerative disease, fatigue syndrome, eating disorder, anorexia nervosa, gastritis, gastric ulcer, Alzheimer's disease, Parkinson's disease, But are not limited to, the above diseases.

The composition of the present invention may contain, in addition to the above-mentioned substances that inhibit the expression, activity and function of 47phox or p67phox, one or more of the active ingredients exhibiting the same or similar functions, or a compound that maintains solubility and / / ≪ / RTI >

The composition of the present invention may also comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, ≪ / RTI >

The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The method of administering the composition of the present invention is not particularly limited thereto, and a method of administering a substance that inhibits at least one of the known expression, activity, and function of 47phox or p67phox can be applied, and parenteral administration For example, it can be administered intravenously, subcutaneously, intraperitoneally or topically, or orally. In the case of parenteral administration, it can be administered through a patch type nasal / respiratory patch attached to the skin. Administration by intramuscular injection may be preferred. The dosage may vary widely depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of the disease.

In another aspect, the present invention provides a method for the treatment of cancer, comprising: (1) providing a cell expressing p47phox or p67phox; (2) a second step of bringing the test substance into contact with the cell; And (3) a third step of screening candidate substances in which at least one of the expression, activity, and function of the p47phox or p67phox is inhibited in cells contacted with the test substance as compared with cells not in contact with the test substance To a method for screening a stress sensitive response modifier.

In the screening method of the present invention, various cells having the characteristics of nerve cells can be used as the cells used in the first step. For example, SH-SY5Y (human neuroblastoma cell line), HT22 But are not limited to, the non-neuronal cell line A549 (human lung adenocassinoma epithelial cell line), KEK 293 (human embryonic kidney-derived cell line), but are not limited thereto. Cells in which p47phox or p67phox are normally expressed and function can be used as such and, if necessary, Such cell lines may be treated with hormones such as glucocorticoids to express, overexpress (compare to untreated cells) and / or activate p47phox and / or p67phox genes, or to express p47phox and / or p67phox genes After transfer into the plasmid, the cells can be contacted with the test substance for a certain period of time, for example 48 hours.

In addition, the cells used in the first step may be provided in the form of an experimental animal. In this case, the screening method of the present invention further comprises the step of applying chronic and / or continuous stress to the experimental animal, Contacting with, including, but not limited to, parenteral or oral administration, stereotactic injection, and the like, one of ordinary skill in the art will be able to select the appropriate method for testing the test substance in the animal. In one embodiment of the invention, the experimental animal may include, but is not limited to, rats, mice, fruit flies, and nematodes. As used herein, chronic / persistent stress includes those defined above. In the case of mice or rats, for example, the methods described in this Example, electroporation treatment, exposure to aggressive mice, cryopreservation, forced swimming, food / water deprivation , Treble sound and strong flicker light exposure, sleep deprivation, daily periodic disturbance, chemical / pain stress treatment with formalin, other drugs, infection control of microorganisms including LPS, isolation, etc. In case of Drosophila and small nematodes Using a reporter gene system that detects ROS increase in the cranial nervous system in the same way as the cultured cell system, candidates can be selected by checking the increase or decrease of ROS after the treatment of the test substance as expression status of the reporter gene. When using an experimental animal model, changes in the activity of p47phox or p67phox will primarily target the brain or nerve cells, but due to the nature of stress hormones such as glucocorticoids (eg cortisone in humans and corticosterone in mice / rats) All organs, tissues, and cells can be investigated because they can act on tissues. In this case too, the increase / decrease of p47phox or p67phox activity by the treatment of the test substance can be measured by the biochemical change of the cranial nervous system, for example, the generation of ROS.

The second step is contacting the test substance with cells expressing p47phox or p67phox.

The test substance includes substances expected to inhibit the expression or activity of p47phox or p67phox as well as inhibition due to the characteristics of the test substance. The test substance is a substance that is expected to specifically inhibit the expression and / or activity of p47phox and / or p67phox or a combination thereof. It may be a low molecular weight compound, a high molecular weight compound, a nucleic acid molecule (e.g. DNA, RNA, Tramadol), proteins, sugars, lipids, and the like. The test material comprises 10 or more bases of single or double stranded nucleic acid, e.g., 10, 11, 12, 14, 15 or more nucleic acid molecules such as siRNA, shRNA or miRNA. The test substance may also be a polypeptide having at least two amino acid residues, such as six, eight, ten, twelve, twenty or fewer, or more than twenty such as fifty (50) amino acid residues.

For the purpose of screening drugs for stress-related diseases, compounds having a therapeutic effect of low molecular weight may be used. For example, compounds having a weight of about 1000 Da such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may constitute a part of a library of compounds, and the number of compounds constituting the library may vary from several tens to several millions. Such a library of compounds can be prepared by reacting peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular weight compounds based on a template, such as benzodiazepines, hydantoins, biaryls, carbocycles, and polycycle compounds such as naphthalene, Carbodihydrate and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely exemplary It is not limited thereto.

The amount of the test substance depends on the specific experimental method to be used and the kind of the test substance, and a person skilled in the art will be able to select an appropriate amount. The candidate substance is a substance that reduces the production of ROS compared to the control group which is not in contact with the test substance as a result of the experiment. About 90% reduction, about 85% reduction, about 80% reduction, about 75% reduction, about 70% reduction, about 65% reduction in the presence of the test substance %, Less than about 60%, about 55%, less than about 50%, less than about 45%, but does not exclude the range. Whether or not the ROS is generated can be detected by various known methods, for example, as described in the examples, and those skilled in the art can select appropriate ones.

When the cell line is used in the second step, contact with the test substance means that the test substance is added to the cell culture medium and the cells are cultured for a certain period of time. Or when the cell is provided in the form of an experimental animal, contact with the test substance is not limited to this, including parenteral or oral administration, stereotactic injection, and those skilled in the art will be able to select the appropriate method It will be possible.

In the third step of the present invention, the amount of p47phox or p67phox expressed in the cells to which the test substance is contacted is compared with the expression level of p47phox or p67phox in the cells not in contact with the test substance. Candidate substances can be selected for biochemical changes of the cranial nervous system by the expression and / or activity increase / decrease of p47phox or p67phox by the treatment of the test substance. In one embodiment, detection of the expression or activity of p47phox or p67phox can be measured by the generation of ROS (Reactive Oxygen Species). ROS detection methods are well known and those skilled in the art will be able to choose the appropriate method and can use, for example, the measurement of lipid peroxidation level, ROS sensitive drugs such as DCF and / or DHE, as described in the examples. As described in the examples herein, a decrease in the production of ROS can be interpreted as a decrease in p47phox or p67phox expression or activity.

Quantitative or semi-quantitative real-time PCR (quantitative or semi-quantitative RT-PCR), quantitative or semi-quantitative RT-PCR, quantitative or semi-quantitative RT- -time PCR), northern blot, and DNA or RNA chip, but the present invention is not limited thereto. In addition, the expression level of 47phox or p67phox protein in the 3-step can be measured by any one method selected from the group consisting of Western blot, tissue immunostaining, and tissue fluorescence staining, but the present invention is not limited thereto.

The step of selecting the test substance which is lower in the expression amount of p47phox or p67phox in the cells to which the test substance is contacted than the amount of p47phox or p67phox expression in the cells not contacted with the test substance as a stress sensitive reaction mitigating agent.

In another aspect, the present invention provides a method for diagnosing a stress disorder caused by a stress-sensitive or chronic stress, which comprises measuring the expression, activity and function of p47phox or p67phox of a biological sample isolated from an individual And a method of providing information.

In one embodiment of the present invention, expression of p47phox or p67phox was increased not only in the brain of the repetitively stressed mouse but also in the blood (Example 5). Based on the above experimental results, the present invention provides a method for diagnosing a stress-sensitive or stress disorder by measuring the expression level of p47phox or p67phox in a subject who is to be diagnosed as a stress disorder caused by stress-sensitive or chronic stress . In the method for providing information for diagnosis, although it is not limited thereto, the blood of the subject to be diagnosed can be collected to confirm the expression level of p47phox or p67phox in the blood. In the above method, when the expression level of p47phox or p67phox in the individual to be diagnosed is increased compared to the level of expression of p47phox or p67phox in the unstressed individual, it is preferable to diagnose the disease as stress-sensitive or stress-disorder.

In the present invention, drugs using the mechanism of inhibiting the expression of p47phox or p67phox can be developed. Drugs screened by the method of the present invention can be used for the treatment of depression, degenerative brain diseases, Can be prevented or treated. In addition, when stress accumulates, the irreversible mechanism proceeds, but if the expression of p47phox or p67phox is well regulated, the mechanism of stress generation can be reversibly controlled.

FIG. 1 shows the result of an experiment using a chronic behavior test to establish a mouse model showing depression and anxiety-like behavior due to chronic stress. (A) shows the effect of imipramine (30 mg / kg) during treatment of repeated cervical stress (RST) (2 hours daily for 14 days, 2h-14d) on 7 week old male C57BL / / day), followed by a series of behavioral tests (SIT, NSF, OF, MBT, TST, FST). (B) shows an example of SIT test method and video tracking results for three groups: control (CON), 2h-14d stressed mice (RST), imipramine administered RST mice (RST + Imi) . (C) is a graphical representation of the results of (B), indicating that the stressed mice spend more time in the corner area than the interaction area. Fig. 1 (D) shows the results of TST and FST showing that immobility of 2h-14d stressed mice (RST) was increased compared to the control group, but that immobility decreased by imipramine treatment. (F) in FIG. 1 (c) shows that the time taken to eat food by the NSF test was increased in RST mice but decreased by imipramine treatment. (G) indicates that the amount of feed consumed for 5 minutes was already increased by the prime treatment. (H) shows that the number of beads concealed by RST mice increased but the number of beads concealed by im- pramine treatment increased as a result of MBT (bead concealing experiment). (I) shows that the motor activity in the central region as a result of OFT was markedly reduced in RST mice and partially restored by imipramine treatment. (J) represents the total cumulative moving distance in the open space and (K) shows the moving distance over time.
FIG. 2 shows that the lipid peroxidation and protein carbonylation were increased in the hippocampus of the brain depending on the stress time and the repetition frequency. (MDA) in the hippocampus of the mice (2h, 4h, 6h) receiving the stress (CON) and stress for a certain period of time (B) and (C) (E) and (F) show the concentration of each malondialdehyde (MDA) in the hippocampus and the concentration of the protein in the hippocampus. The degree of protein carbonylation is shown.
FIG. 3 is a graph showing the increase in ROS stress depending on the dose and duration of glucocorticoid, which is a stress hormone, in SH-SY5Y cells. (A) shows the process of treatment of SH-SY5Y cells with glucocorticoid (CORT: 400 or 800 ng / ml) for 2 hours, (B) shows the MDA concentration (expressed in% of the 24h control) in SH-SY5Y cells, and (C) and (D) show the experimental design of SH-SY5Y cells (10 μM) and DHE (10 μM) to detect the ROS of the cells, and the graph below shows the amount of fluorescence. CON: control cells; CORT: glucocorticoid treated cells (800 ng / ml, 48 hours); Lower panel: High resolution, scale bar: 20μm.
FIG. 4A is a graph showing a change in the expression pattern of genes regulating ROS concentration in the mouse brain due to repetitive stress. (A) and (B) show the expression of genes regulating ROS concentration in stressed mice (2h-14d) and control prefrontal cortex (PFCX; A) and hippocampus (HP; B) (C) of FIG. 4b schematically shows the composition of NADPH oxidase, and (D) shows that the expression of NADPH oxidase subunit was upregulated due to repeated physical stress. As a result, RT-PCR results for NOX2 subunits (p91phox, p22phox, p47phox, p67phox, and p40phox) and beta actin at the PFCX and HP sites of mouse (2h-14d) (R)
Figure 5 shows that the expression of p47phox and p67phox can be up-regulated in the blood of mice subjected to repetitive cramping stress. Data were expressed as mean ± standard error (n = 4-6). ** indicates a significant difference in p <0.01 between the indicated groups (Student t-test).
Figure 6 shows that in SH-SY5Y cells CORT-induced ROS production was blocked by NADPH oxidase inhibitor treatment. 6a shows the increase in ROS concentration in SH-SY5Y cells treated with glucocorticoids (400 ng / ml) for 48 hours in aposinin (Apo, 500 μM), RU486 (RU, 20 μM), SOD mimetics MnTBAP (Mn, 100 μM) , Or vitamin C (Vit C, 200 [mu] M) was observed with DCF (10 [mu] M) staining and expressed as a percentage of the control. Figure 6b shows that apoptinin blocks the NADPH oxidase subunit p47phox from being activated by glucocorticoids and migrating to the cell membrane, resulting in an untreated control, SH-treated for 48 hours with glucocorticoid (400 ng / ml) SY5Y cells and treated apocrine-treated cells were observed with anti-p47phox antibody and DAPI staining.
Figure 7 shows the results of the up-regulation of p47phox expression by glucocorticoid treatment in SH-SY5Y cells. (A) was the result of observation of p47phox expression in SH-SY5Y cells treated with glucocorticoid (400 ng / ml) or H ₂ O ₂ (200 μM) for 24 hours by real-time quantitative RNA analysis and each sample was expressed as a multiple of the control group. (B) and DCF (B) show that ROS production induced by glucocorticoid (400 ng / ml) was completely blocked by treatment with cyclohexamide (CHX; 1 or 10 μM) C). Each value was expressed as% of 24h control group.
Fig. 8 shows the results of showing that glucocorticoid-induced ROS production was blocked when the expression of p47phox gene was reduced using siRNA in SH-SY5Y cells. (B) and (C) show that SH-SY5Y cells are transferred to siRNA (50 or 100 μM), while siRNA (A) (B) and DHE (C) after 72 hours of treatment with glucocorticoid (400 ng / ml).
FIG. 9 shows that administration of apoptinin, an NADPH oxidase inhibitor, to mice inhibited ROS production due to stress in the brain. (A) represents the experimental design and shows that imipramine (Imi; 20 mg / kg), aposinin (Apo; 15 mg / kg) or vitamin C (Vit C: 15 mg / kg) was administered intraperitoneally. (C) and (D) show the results of the behavioral tests TST (C) and FST (D) by each drug administration.
Figure 10 shows the results of blocking depressive behavior due to chronic stress when knockdown of p47phox expression in the brain is blocked. (A) shows the expression of GFP in HeLa and A549 cells at 8 hours after infection with Lenti-p47phox-shRNA expressing GFP. (B) Lenti-p47phox-shRNA (also expressing GFP) or Lenti-GFP, and shows the period of stress and the timing of the test. (C) and (D) show the TST (C) and FST (D) test results in the stressed mouse (RST) and control (CON) mice when infected with each of the viruses.
Figure 11 shows that mice with only one copy of p47phox (p47phox +/-) (2h-14d stress) and depression-like behavior due to chronic stress (2h-14d stress). (A) shows the genotype of a mouse with only two copies (+ / +) of p47phox gene (gel photograph lanes 2-3) and only one copy (gel photograph 4-5 lanes) And then analyzed by MspI restriction enzyme digestion. M: marker; 100 bp ladder, lanes 3 and 4 show that additional bands were generated by p47phox gene deletion. (B) added 2h-14d stress to p47phox heterozygote mouse (+/-) and wild type (+ / +) mice in experimental design. (C) and (D) are the results of TST (C) and FST (D) performed the day after the last stress treatment.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Materials and Methods

animal

Seven week old male C57BL6 mice were purchased from BioLink. The mice were housed in a cage for two weeks before the experiment in an environment where humidity and temperature were controlled. All animals were handled according to the guidelines for laboratory animal management at Ewha Womans University Medical School. P47phox genetic mouse (+/-) was as described in Huang et al., 2000 and purchased from Jackson Laboratories (USA). Genotyping of the genetically-modified mouse was carried out by separating the genomic DNA from the tail of the mouse and using the following primers: 5-TGG AAG AAG CTG AGA GTT GAG G-3 (SEQ ID NO: 1), 5-TCC AGG AGC TTA TGA ATG ACC-3 (SEQ ID NO: 2). Amplified by PCR and digested with MspI and analyzed by agarose gel electrophoresis.

Restraint Stress

In the present study, we conducted a series of experiments to determine the effects of cognitive stress. Briefly, approximately 20 to 23 grams of 7 week-old male C57BL / 6 mice were individually placed into a 50 ml polypropylene conical tube that was individually ventilated, capped with a medium sized tube approximately 3 cm long, And the mouse was completely detached in this apparatus. These detention tests started at a fixed time (10 am) and were performed for 2 hours a day and repeated for 14 days. After the custody experiment, the mice were housed in a cage and kept in a normal environment. Water and feed were freely consumed.

drug injection

Two hours of confinement One hour before the start of the confinement, the drug was injected intraperitoneally into the mice of interest and this procedure was repeated during the custody experiment. The name, source, and route of administration of the drugs used were as follows: Imipramine hydrochloride (Sigma (St. Louis, MO)) at a dose of 20 mg / kg (ip); Apocynin (Sigma), administered 15 mg / kg (i.p.); Vitamin C (L-Ascorbic acid, Sigma), dose 15 mg / kg (i.p.). Aposinin was dissolved in DMSO, diluted with saline, and the other drug dissolved in saline. Finally, each drug diluted with 100 [mu] l of 0.9% saline was administered according to the above-mentioned administration method and dose.

In order to exclude the occurrence of stress due to injection for the behavioral experiment, laboratory chow or laboratory feed containing 30 mg / kg / day imipramine was also used independently of the above case. These feeds were prepared by milling general laboratory feeds, mixing imipramine, shaping and irradiating gamma rays. The feed used as a control was also prepared with the exception of the imatinam (Seo et al. 2010).

Behavioral assessment experiment

The mice were transferred to the test room 30 minutes before each behavioral assessment test and allowed to stay in the same room throughout the test. The test was carried out as described in Figure IA (A). Behavioral assessment was performed using a computer video-tracking system (SMART; Panlab SI, Barcelona, Spain) and analyzed one hour after the last injection. At all times, the sound was blocked by 55-60 dB white noise (noise).

Social Interaction Test (SIT)

SIT was performed essentially as described previously except for a few variations (Berton et al., 2006). Each test subject's mouse was placed in the open field and traced twice in succession (5 min each time, optimal sensitivity / throughput ratio). For the first session (non-target condition), the mouse to be examined was placed in an open space with an empty plexiglass mesh cage (15 x 8 x 17 cm) without the target animal to accommodate. The second session (target condition) was the same as in the first session, but the social interaction target animal (aggressive B6 / SJL male mice, aged at least more than the test animal) was placed in the open space with the Plexiglas mesh cage, . Between the two sessions, the subject mouse was moved from the open field to the home cage and left for about one minute. For each non-target and target session, the video tracking data was used to calculate the time spent by the subject mouse in the interaction area (8 cm wide area around the target animal cage) and the corner area (18 x 18 cm) opposite the cage (In seconds). Mobility was measured in the absence of the target animal B6 / SJL mouse for 5 minutes followed by a 5 minute hold with the target animal in the cage. The measurements are obtained using SMART software.

Tail hanging test ( tail suspension test ; TST )

The tail of the mouse to be investigated was fixed to a metal rod located 50 cm above the table surface, and the mouse was suspended and immobilized for 6 minutes. The experiment was carried out in a noiseless room and a safety mat was placed on the table (Steru et al., 1985, Bilkei-Gorzo et al., 2002).

Forced swimming inspection ( forced swim test ; FST )

The mice were placed in a plexiglass cylinder (height: 27 cm, diameter: 15 cm) at a depth of 14 cm and filled with water at 24 ° C, and allowed to preliminarily swim for 15 minutes under conditions of escaping or not touching the floor. After 15 minutes, I took out the mouse, wiped it with a towel, and sent it back to the home cage. After a day, mice were forced to swim for 6 minutes. The mice were allowed to adapt for the first minute, and behavior was observed for the next 5 minutes. Immobility time is defined as the sum of the hours the animal moves without movement of all limbs. Detailed recording methods are described in the reference (Kim et al., 2006).

Feeding in a new environment Inhibitory  inspection( Novelty Suppressed Feeding , NSF )

This experiment measures the anxiety caused by stress, and measures the time that an animal hesitates until the animal first consumes the food it consumes in an unfriendly environment. This test is very sensitive to the pharmacological test of chronic depression. Before the test, the feed was removed from the cage for ~ 24 hours and the water was allowed to drink arbitrarily. At the end of this period, a 2 x 2.5 cm oval feed pellet was placed on a circular white filter paper (diameter 150 mm) and centered in the open field (45 x 45 x 40 cm). Each mouse was removed from the home cage, placed in an air cage for 30 minutes without water and feed, and placed in an open space illuminating 1400 lux. From this time on, the dietary and home cages consumed for 5 minutes were recorded. As soon as the mice started to feed, the animals were transferred to a home cage and fed a pre-measured weight of feed for 5 minutes. At the end of 5 minutes, the weight of the feed put into the cage was measured and the amount of feed consumed was calculated.

Beading test ( Marble Burying Test , MBT )

The bead concealment test was conducted to measure anxiety and was basically performed according to the existing method (Deacon, 2006, de Kloet ER et al, 2005), except for some modifications. To summarize, the rug is filled in the empty home cage to a height of 5 cm from the floor, 12 balls are placed in a grid, and the mouse is released and left free for 30 minutes. After that, the number of hidden beads was counted. If the beads were <25% visible, they were considered hidden. Mice pre-dosed with antidepressants were less cocoon-hungry compared to control mice.

Open Space Test ( Open Field , OF )

The test animals were placed in the center of the open space of a white plexiglass chamber (45 x 45 x 40; 70 lux) and the mobility was measured for a fixed time. The horizontal mobility was determined by the distance traveled by the test animals. The inner rectangular area occupying 30% of the open space is defined as the central part.

Measurement of lipid peroxidation

Lipid peroxidation was assessed by measuring the level of malondialdehyde (MDA) using the Bioxytech MDA-586 kit, as described previously (Im et al., 2006; Seo et al., 2010a-c; 2011a-b) Respectively.

Briefly, brain tissue was homogenized with 4 times volume of ice-cold 20 mM PBS (pH 7.4) containing 5 mM butylated hydroxytoluene. The homogenate was then centrifuged at 3,000 g for 10 minutes at 4 ° C, and the supernatant was used for each assay. To each reaction, 10 μl of probucol and 640 μl of diluted R1 reagent (1: 3 methanol: N-methyl-2-phenylindole) were added and mixed with 150 μl of 12N HCl. After reacting at 45 ° C for 60 minutes, the cells were centrifuged at 10,000 g for 10 minutes. The supernatant was separated and absorbance was measured at 586 nm. MDA data were normalized to protein concentration and values were expressed as a percentage of the control (sham control). Protein-bound MDA content was obtained by subtraction.

Determination of protein carbonylation content

Protein carbonyl content was determined by the method of Lee et al. (2009). The supernatant (500 μl) of the brain homogenate was precipitated with 500 μl of 20% trichloroacetic acid (TCA). After centrifugation at 10,000 g for 15 minutes, the pellet was resuspended at 50 &lt; 0 &gt; C with 0.5 ml of 2 mM HCl, 10 mM 2,4-dinitrophenylhydrazine. The reaction was allowed to proceed for one hour at room temperature in the dark room with periodic stirring every 10 minutes, after which the sample was precipitated with 0.5 ml of 20% TCA. This procedure was repeated three times using 10% TCA. The resulting precipitate was dissolved in 2 mM NaOH at 37 &lt; 0 &gt; C. The protein carbonyl content was determined by measuring the absorbance at 360 nm and expressed in units of nmol / mg protein.

Blood separation

Mouse blood cells were prepared from the blood collected from the abdominal aorta of anesthetized rats in a 1 ml disposable plastic syringe. Whole blood was collected in a tube and centrifuged at 1,500 rpm for 15 minutes at room temperature. The supernatant and pellet obtained were referred to as serum and blood cells, respectively.

real time PCR On by mRNA Quantification of

RNA sample preparation and real-time PCR were performed as previously described (Ha et al., 2008). For RNA quantification, the superior prefrontal cortex, hippocampus, and blood cells of chronic RTS-treated mice were collected. Total RNA was purified using Trizol (Invitrogen Carlsbad, CA, USA) reagent and treated with DNase I to prevent genetic contamination. Reverse transcription was performed using a reverse transcription system (Promega, Madison, Wis., USA) according to the manufacturer's instructions. First-strand cDNA was synthesized with 1 μg total RNA at a total volume of 20 μl using the Promega RT PCR kit.

The PCR reaction was carried out in a final volume of 10 μl containing 2 μl of 2 × iQ SYBR Green supermix (Bio-Rad, Richmond, Calif., USA), 1 μl each of 5 pmol / μl of forward and reverse primers and 5 μl cDNA 20 μl using a CFX 96 real-time PCR System Detector The reaction cycle was as follows: 5 min at 95 ° C, 20 sec at 95 ° C, 20 sec at 56 ° C , And for 20 sec at 72 ° C for 40 sec. The specificity of the amplification is determined by the DNA melting curve for autofluorescence data loaded from 55 ° C to 95 ° C using graduated temperature increase (0.5 ° C) The difference in amplification factor was determined by using embedded software called Gene Expression Analysis for Bio-Rad CFX Manager and real-time PCR of the target gene against GAPDH and β-actin used for reference Based on the amplification, The real time PCR analysis was performed using real time PCR as described in Table 1 (Real Time PCR Primer Set (Mice: SEQ ID NOS: 3 to 28) and 2 (Real Time PCR Primer Set (Human: SEQ ID NOS: 3-6, SEQ ID Nos. 29 to 32) PCR primer set. Real-time PCR was performed 6 times for each group and each site.

RT - PCR Using cDNA  Concentration measurement

The amount of cDNA was measured using real-time PCR. The primers used for amplification of specific sites of the transcripts measured in the experiment are as follows:

gp91 - phox , 5'-GGTTTATGATGATGGGCCTAA-3 '(SEQ ID NO: 33) and 5'-GCACTGGAACCCCTGAGAAA-3' (SEQ ID NO: 34); p22 - phox , 5'-CCCCGGGGAAAGAGGAAAAA-3 '(SEQ ID NO: 35) and 5'-AGGACAGCCCGGACGTAGTA-3' (SEQ ID NO: 36); p67 - phox, 5'-GGGAACCAGCTGATAGACTA- 3 '( SEQ ID NO: 37) and 5'-TCCATTCCTCYTTCTTGGCA-3' (SEQ ID NO: 38); p47 - phox , 5'-CAGCCAGCACTATGTGTACA-3 '(SEQ ID NO: 39) and 5'-GAACTCGTAGATCTCGGTGAA-3' (SEQ ID NO: 40); And p-40- phox , 5'-TGGCCCAGCAGCTGCG-3 '(SEQ ID NO: 41) and 5'-CGRTAGCGGCGGTAGATGAG-3' (SEQ ID NO: 42). gp91-phox, p22-phox, p40-phox, p47-phox and p67-phox primers were used as described previously (Tammariello et al., 2000). GAPDH and beta-actin were used as an internal control. The PCR reaction was performed three times independently.

Lentivirus  Vector containing ( Lentiviral vector ) And concentration

Recombinant lentiviruses were produced as described previously (Min et al., 2010). Briefly, three plasmids containing the transgene vector (Lenti-p47phox-shRNA, purchased from OpenBiosystems, RMM4431-98723759), membrane glycoprotein lentivirus vector (Macrogene., Korea) and packaging vector were ligated into 293T cells (Invitrogen Corp., CA, USA) at a molar ratio of 1: 1: 1. The culture supernatant containing the viral vector particles was injected intranuclearly, collected 48 hours, filtered through a 0.45 μm membrane and stored in a -70 ° C freezer. The extent of lentiviral vector expression was analyzed by RT-PCR, Western blot, and ELISA. Titer (titration) was determined by p24 ELISA after infection with HeLA cells. The value thus obtained was about 10 ⁶ -10 ⁷ transduction unit (TU) / ml. GFP expression was confirmed after intranuclear injection into HeLA or A594 cells and photographed using a fluorescence microscope (Nikone TE2000U, Japan). Virus was filtered with Centricon (Millipore Corp., Billerica, MA, USA). The viral particles were further concentrated by ultracentrifugation at 80,000 g for 2 hours, and this concentration process was repeated twice. Finally, it was obtained in an amount of about 10 ¹⁰ -10 ¹² TU / ml.

Stereotaxic injection of lentivirus-shRNA

Male C57BL / 6 mice (20-25 g) were anesthetized by intraperitoneal injection of a 3.5: 1 mixture of ketamine (50 mg / ml) and xylazine hydrochloride (23.3 mg / ml) , And placed in a stereotactic device as described in previous studies (Kim and Han, 2006). A 3-L volume of lentil shRNA was injected into the right lateral ventricle with 0.5 g / min of a 28-G needle at the rate of 0.5 l / min in the right lateral ventricle (in millimeters relative to the bregma) anterior-posterior, -2.4; midlateral, 2.0; dorsal-ventral, -2.1) (Franklin and Paxinos, 1997). After 5 minutes, the needle was removed by 3 steps for 3 minutes to minimize the reflux, and the mouse was kept on a warm pad so as not to break. Anesthetized mice were placed in a home cage until used for analysis. After 14 days of injection, depression behavior test was performed. shRNA control; Mouse pGIPZ lentivirus shRNXmir (RMM4431), Lenti shRNA-p47phox (RMM4431-98723759, V2LMM_3405) were purchased from Open Biosystems, Inc. (USA).

Tissue immunochemistry

The brains were fixed with 4% paraformaldehyde under gentle perfusion and fixed in the same solution overnight at 4 ° C. As described in previous studies (Lee et al., 2009), brains were cut at 40 μm intervals using a vibratome to prepare brain slices. After reacting with anti-p47phox antibody (Santa Cruz, USA), anti-HNE (HNE11-S) antibody (Alpha Diagnostic Intl. Inc, USA) as primary antibody, Alexa Fluor 488- conjugated antibody or rabbit IgG Was visualized using a second antibody coupled to Texas Red 594-conjugated antibody (Invitrogen Corp., USA) and FITC (Fluorescein isothiocyanate) and TRITC (Jackson ImmunoResearch, USA) Nylated HRP (Vector Laboratories, USA) was used as directed by the manufacturer. After the color development, the vector shields (Vector Laboratories) were placed on the slides and observed with a microscope and analyzed with the built-in camera and software (Olympus BX 51 microscope equipped with a DP71 camera and DP-manager and DP-Controller software, Olympus Co., Japan ). The number of cells was measured by a blinded method using a TOMORO ScopeEye 3.6 program (Techsan Community, Korea) after the image of the stained area was converted into a digital image.

Cell culture and drug treatment

Cell culture of SH-SY5Y neuroblastoma cell line has been described in previous studies (Seo et al., 2010b-c; 2011a). Briefly, SH-SY5Y cells were incubated in a wet incubator (95%) with Dulbeccos modified Eagles medium (DMEM) containing 10% heat-inactivated FBS, penicillin (20 units / ml) and streptomycin Air and 5% carbon dioxide). When the cells grew to confluence 70-80% of the culture dish, they were used in the experiment. Glucocorticoid (CORT), H ₂ O ₂ . RU486 (mifepristone), MnTBAP, apocyanin and vitamin C were purchased from Sigma. CORT, H ₂ O ₂ , MnTBAP, and vitamin C were dissolved in PBS at a concentration of 100 μM. Aposinin and RU486 were dissolved in DMSO at a concentration of 100 μM and then diluted with PBS. Glucocorticoid and H ₂ O ₂ were used for cells cultured in serum-free DMEM.

Transfers and transfers siRNA  ( Small Interference RNA )

In order to knock down p47phox, SH-SY5Y cells were seeded 24 hours before transfer to allow confluence of 70% of the recipient culture dish. siRNA (siRNA-p47phox) was synthesized in Bioni (Korea). SiRNA-p47phox sequence of sense strand: 5'-CUA UUU CCC AUC CAU GUA U-3 '(SEQ ID NO: 43); And antisense strand: 5'-AUA CAU GGA UGG GAA AUA G-3 '(SEQ ID NO: 44). Four deoxyribosucleotide overhangs (dTdT) were added to the 3 'end of each siRNA strand. The oligonucleotides siGLO Green [D-001630-0105] and [D-001210-0205] labeled with the non-target control siRNA 6-carboxyfluorescein fluorophor were purchased from Dharmacon Inc (USA). Cells were transferred using a Lipofectamine reagent (Invitrogen) at a concentration of 100 nM siRNA diluted in DMEM as directed by the manufacturer. p47phox depression was confirmed by immunocytochemistry using anti-p47phox antibody.

ROS measurement in cultured cells

SH-SY5Y cells in 96-well plates were incubated in a medium containing 10 [mu] M DCF for 30 minutes at 37 [deg.] C. ROS was visualized using CM-H2DCFDA [5- (and-6) -chloromethyl-2,7-dichlorodihydrofluorescein diacetate], a transmembrane fluorescent dye sensitive to hydrogen peroxide-related ROS as described in the prior art (Im et al. , 2006; Seo et al., 2011a).

Briefly, SH-SY5Y cells were washed with HEPES buffer (120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl ₂ , 1.8 mM CaCl ₂ , 20 mM HEPES, 10 mM NaOH, 15 mM glucose) for fluorescence level analysis. Fluorescence levels were determined by excitation at 488 nm and emission at 510 nm for DCF using excitation at 540 nm and emission at 590 nm for DHE using a spectrophotometer (SpectraMaxM5, Molecular Devices Corporation, Sunnyvale, CA. USA) . The average value was measured three times and used as one piece of data. The stained images were analyzed using a DP71 camera and an Olympus BX 51 microscope (Olympus Co.) equipped with DP-manager and DP-Controller software. CONFOCAL images were analyzed with a Zeiss LSM510 / META / NLO laser scanning microscope and Zeiss Plan-Neofluar software (Carl Zeiss, USA). The number of cells was measured by a blinded method using a TOMORO ScopeEye 3.6 program (Techsan Community, Korea) after the image of the stained area was converted into a digital image. Fluorescence of each sample was indicated by a bar graph of percent of sham in 24h using a bar graph.

Immunocytochemistry

The cells were fixed at room temperature with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10 minutes, permeabilized with 0.2% Triton X-100 in 0.1 M PBS for 10 minutes, For a period of time. After blocking, the cells were incubated overnight at room temperature in a 3% BSA solution containing anti-p47phox antibody (1: 1000). Cells were washed with 0.1 M PBS and incubated with fluorescein-conjugated secondary antibody (TRITC, 1: 100, Sigma) for 1 h, then washed and stained with fluorescein-binding solution (DAKO, Denmark) Minute mounting. The dyed images were then captured / analyzed using a DP71 camera and an Olympus BX 51 microscope equipped with DP-manager and DP-Controller software (Olympus Co.)

Statistical processing

Statistical analysis was performed using GraphPad PRISM software 4.0 (GraphPad Software Inc., USA). Statistical analysis was performed using ANOVA and Newman-Keuls multiple test method. All data were mean ± SEM, and a statistical difference of 5% was used unless otherwise stated.

Example  1: Continuous depression-related behavior due to repetitive custodial stress

To investigate the effects of chronic stress such as repetitive cramping stress (2h-14d, see Experimental Method), experiments were conducted with the contents shown in (A) of FIG. 1a. The specific experimental method was described in detail in the experimental method.

2h-14d The social interactions performed on the 15th day of detention use a video tracking system (Figs. 1A and 1B). As a result, the control mice were close to or interacted with the target mice for 125 seconds during the observation period of 300 seconds, The mice to which the mice were administered decreased the time spent with the target mice and spent time in the corner areas (Fig. 1A, C). On the other hand, there was no difference between the control group and the test group in the absence of the target mouse. To confirm whether these persistent non-social behaviors are similar to stress-induced depression in humans, imipramine, widely used as an antidepressant drug, was administered to prison mice and the same tests as above were performed. Immediate administration of imipramine for 15 days after the last imprisonment stress increased the time spent in the interaction area, and at the same time the time spent in the corner area decreased (Fig. 1a, C). These results indicate that the symptoms induced by detention are improved by the drug and that the induced symptoms are due to depression.

The results of social interaction are also verified in the various behavioral tests mentioned below. In the case of TST (FIG. 1B, D), TST was performed on the 16th day after 2h-14d pricking stress, and the immobility time was significantly increased (p <0.01) in the stressed mice as compared with the control. These symptoms were not observed with imipramine for 15 days after 2h-14d prison stress, suggesting that the symptoms are related to depression. FST (Fig. 1B, E) showed the same result. FST was performed 17 days after 2h-14d pricking stress, and the immobility time was significantly higher in the stressed mice than in the control group (p < 0.01). On the other hand, these symptoms were not observed with imramine for 16 days after 2h-14d prison stress, suggesting that the symptoms are related to depression. These results indicate that 2h-14d prison stress can lead to sustained depressive behavior that can be alleviated by imipramine, an anti-depressant.

 Stress-related depression has been found to be closely related to persistent anxiety-related behavioral changes (Kim and Han, 2006). Therefore, it was investigated whether 2h-14d prison stress caused this phenomenon. The NSF test was conducted to measure the hesitation time of food intake under the new environment. The NSF test performed on the 16th day after 2h-14d stress showed that the time taken to feed the mice was much longer (P <0.01), indicating that custodial stress results in anxiety disorder, which lasts for up to 17 days after stress. These anxiety disorders recovered to the control level when the antidepressant imipramine was administered for 17 days after stressing (Fig. 1C, F and G).

MBT was performed with other behavioral tests related to anxiety. MBT was performed 18 days after 2h-14d stress, and the stressed mice had a significantly lower number of hidden dictation compared to the control group, indicating that the mice were unstable. In this case also, when imipramine was administered, the MBT results were restored to the control level (Fig. 1C, H). In the open field test, the stressed mice had less movement distance on the central part of the open field than the control group, indicating anxiety symptoms, and this behavior was partially restored by the administration of the primeam (Fig. 1C, I). There was no difference in total travel distance in the open field (Fig. 1C, J and K). Taken together, these results indicate that depression and anxiety behaviors have been sustained due to custodial stress. In FIG. 1, data are shown as means ± SEM (n = 20-22), and * and ** indicate p <0.05 and p <0.01, respectively. One-Way ANOVA <Newman-Keuls multiple comparison test was used.

Example 2: ROS (Reactive Oxygen Species) in the brain due to cramping stress increase

The present inventors have examined whether the stress-induced continuous depression-related behavior is associated with the brain's ROS regulation system.

Repeated stress leads to high oxidative stress in the brain through the glucocorticoid, the stress hormone in the blood. However, the mechanism by which glucocorticoids increase ROS and whether ROS causes depression and anxiety-like behavior are not known.

To elucidate the mechanism of ROS generation by glucocorticoids, we first examined the lipid peroxidation and protein carbonylation as indicators of oxidative stress in the brains of mice exposed to cortical stress for different periods of time. 2 hours, 4 hours, and 6 hours (FIG. 2A), and the control stress was divided into groups performed for 2 hours each day for 1, 3, 5.7, 10, or 14 days 2D).

The degree of malondialdehyde (MDA) and protein carbonylation as a result of lipid peroxidation in the hippocampus was increased depending on stressed time (B, C) and repetition frequency (E, F) ROS increased significantly after.

In FIG. 2, data are shown as means ± SEM (n = 4-8), and * and ** indicate p <0.05 and p <0.01, respectively. One-Way ANOVA <Newman-Keuls multiple comparison test was used.

Example 3: Concentration of SH-SY5Y cells to glucocorticoids and induction of time-dependent increase in ROS concentration

To elucidate the mechanism of ROS production by glucocorticoids, SH-SY5Y human neuroblastoma cells were treated with a glucocorticoid (CORT) and the production of ROS was investigated. Experimental designs with different doses and times of glucocorticoid administered are described in FIG. 3, A. Specifically, the process of treating SH-SY5Y cells with a low concentration of glucocorticoid (400 ng / ml or 1.15 μM) or a high concentration (800 nm / ml) for 2 hours, Repeated experiments were performed.

As a result, MDA concentration was increased depending on the amount of glucocorticoid and treatment period as shown in FIG. 3 and FIG. In order to identify the factors involved, glucocorticoid-treated cells were stained with DCF, a sensitive dye for hydrogen peroxide, and DHE, a peroxide anion-specific stain, and then observed with a microscope and a spectrophotometer. The results are shown in Figures 3, C and D, respectively. High DCF-reactive fluorescence was detected in SH-SY5Y cells, which had been subjected to repeated treatment for 3 days with a high concentration of glucocorticoid as compared with the untreated control group. These results indicate that the concentration and time of glucocorticoid increase ROS and peroxide in the cells. In FIG. 3, data are shown as mean ± SEM, with * and ** representing p <0.05 and p <0.01, respectively, indicating significant differences between the control and experimental groups. One-Way ANOVA <Newman-Keuls multiple comparison test was used.

Example 4: Increased expression of NADPH oxidase in the brain due to chronic stress

As mentioned earlier, custodial stress induced long-term depression and anxiety-like behavior in mice and resulted in an increase in ROS concentration in the brain. In this experiment, intracellular factors involved were identified. Factors known to be important for ROS generation were examined by real - time PCR in brain tissues of detained and untreated control mice for factor analysis. The genes examined include peroxiredoxin (PRX1), cytoplasmic Cu / Zn superoxide dismutase (SOD1), catalase (CAT), hemoxigenase- 1 (heme oxygenase-1 (HO-1)), p47phox, p67phox, cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS) (NF-κB) , tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β). The results are shown in FIG. 4, A (prefrontal cortex, prefrontal cortex, PFCX) and B (hippocampus, HP). Expression patterns of the investigated genes were different in PFCX and HP. Especially, the expression levels of p47phox and p67phox, which are NADPH oxidase subunits, in the stressed mice were significantly increased compared to the control group. Data are presented as mean ± SEM (n = 6-9), and * and ** indicate significant differences between groups with p <0.05 and p <0.01, respectively (Student's t-test).

NADPH oxidase includes the subunits p47phox, p67phox, p40phox, gp91phox (NOX2), and p22phox with the "phox" NOX2 and p22phox are located in the cell membrane, and p47phox is present in the cytoplasm. When it is phosphorylated, it moves to the membrane together with p67phox and p40phox to form an active NOX2 complex. The activated complex carries out the exchange of Rac GDP with GTP, which in turn transfers electrons from NADPH to oxygen and produces H ⁺ and peroxides as by-products. The reaction equation is as follows: NADPH + 2O ₂ ↔ NADP ⁺ + 2O ₂ ^{- -} + H ^+. (Fig. 4, C).

The expression of p47phox and p67phox in the HP and PFCX of mice subjected to cortical stress by 5 kinds of subunits constituting NADPH oxidase was increased by RT-PCR (Fig. 4, D). These results are consistent with the results observed in FIGS. 4A and 4B, indicating that NADPH oxidase is involved in the generation of ROS due to chronic stress. In order to elucidate the detailed mechanism, NADPH oxidase activation by p47phox phosphorylation and membrane transduction was investigated. In Figure 4 the data are expressed as means ± SEM (n = 6-9), and * and ** indicate significant differences between groups with p <0.05 and p <0.01, respectively (Student's t-test).

Example 5: Increased Expression of p47phox and p67phox in the Blood of Chronic Stressed Mice

We examined whether repetitive stress increases the expression of p47phox and p67phox in the brain as well as in the blood of mice.

Mouse blood cells were obtained from the blood of mice subjected to repeated cervical stress (2h-14d) and non-cervical stress, and the degree of expression of p47phox and p67phox in the blood was confirmed by real-time PCR.

As a result, it was confirmed that the expression of p47phox and p67phox was significantly increased in the blood of mice subjected to repetitive cramping stress, compared with the blood of mice not subjected to custodial stress (FIG. 5).

From the above results, repetitive stress not only increased the expression of p47phox and p67phox in the brain mitochondria, but also increased the expression of p47phox and p67phox in blood cells, and the blood was collected from the blood cells to measure the expression of p47phox or p67phox, And depressive symptoms due to repeated stress can be diagnosed. In addition, by suppressing the decrease of the expression of p47phox or p67phox in the blood, or by promoting its activity, it is possible to alleviate the stress sensitive response and to treat the stress disorder caused by chronic stress.

Example  6: NADPH Oxydase  Gt; glucocorticoid &lt; / RTI &gt; by up-expression SH - SY5Y  In the cell ROS  increase.

After SH-SY5Y cells were treated with glucocorticoids, ROS production was examined by DCF staining in the presence and absence of apoptinin, one of the NADPH oxidase inhibitors. The results are shown in FIGS. 6A and 6B. It can be seen that increased ROS by glucocorticoids was completely blocked by treatment with aposinin (Apo, 500 [mu] M). ROS synergy was also blocked by the glucocorticoid receptor antagonist RU486 (RU, 20 μM), SOD mimetics MnTBAP (Mn, 100 μM), and vitamin C (Vit C, 200 μM). Data are presented as means ± SEM (n = 6), with * and ** representing significant differences between the two groups, p <0.05 and p <0.01, respectively. One-way ANOVA and Newman-Keuls multiple comparison tests were used.

These results indicate that the activity of NADPH oxidase is an important factor in the production of glucocorticoid-induced ROS.

In order to investigate the role of p47phox in the NADPH oxidase activity, the transfer of p47phox to the membrane (Suh et al., 2007; 2008), which is a necessary preliminary step of activation, was performed in SH-SY5Y cells treated with glucocorticoid Were used for the study. The results are shown in FIG. 6B. In the case of untreated cells, p47phox remained in the cytoplasm, but when treated with glucocorticoid, it was found to have migrated to the cell membrane, and this phenomenon disappeared by treatment with apocyanin.

These results demonstrate that NADPH oxidase mediated increased ROS by glucocorticoids.

Cells were treated with 200 μM hydrogen peroxide to determine whether p47phox increase by glucocorticoid was indirectly due to ROS. As a result, there was no change in the amount of p47phox expression, indicating that the increase in p47phox expression was independent of ROS (Fig. 7A). In FIG. 6, data are shown as mean ± SEM (n = 5-6), and * indicates significant difference compared to CORT alone treatment group at p <0.05. One-way ANOVA and Newman-Keuls multiple comparison tests were used.

We investigated whether the increase of ROS by glucocorticoids requires new protein synthesis or glucocorticoid metabolism. For this purpose, cyclohexamide (CHX), which is a protein synthesis inhibitor, was treated in the course of glucocorticoid treatment. ROS was stained with DCF and DHE, which are sensitive dyes, and ROS and peroxide formation were completely blocked (FIGS. 7, B and C ).

These results imply that the synthesis of NADPH oxidase subunits is required for the production of ROS and peroxides in SH-SY5Y cells treated with glucocorticoids. These results also indicate that long-term (> 24h) glucocorticoid treatment is required for the production of ROS and peroxides in SH-SY5Y cells (Fig. 3), and that chronic stress is required to induce high oxidative stress in mice 2). &Lt; / RTI &gt;

To confirm the association of increased p47phox with glucocorticoids and increased ROS and peroxide production, p47phox expression was down regulated using p47phox-siRNA. The results are shown in Fig. SH-SY5Y cells were transfected into si-GFP (at a concentration of 100 μM siRNA, 87.32 ± 1.73% or more were GFP-positive), and siRNA was delivered to almost all cells (FIG. SH-SY5Y cells were transferred to p47phox-siRNA 72 hours later, cells were treated with glucocorticoid (400 ng / ml) for 48 hours and ROS was analyzed by DCF and DHE staining. ROS production induced by glucocorticoid (FIG. 8, B) and peroxide production (FIG. 8, C) in cells transfected with p47phox-siRNA (50 or 100 μM) decreased dependent on the amount of siRNA treated , Which indicates that up-regulation of up-regulation of p47phox is a major factor in the production of ROS and peroxide by glucocorticoids. In Figure 8 the data are expressed as mean ± SEM (n = 4-6), with * and ** representing significant differences between the two groups, p <0.05 and p <0.01, respectively. One-way ANOVA and Newman-Keuls multiple comparison tests were used.

Example  7: p47phox  Behavior control in response to chronic custodial stress

In order to understand stress - induced up - regulation of p47phox in brain, we investigated the relationship between p47phox activity and ROS production and depressive behavior in hippocampus of brain. The experimental design is illustrated in Figure 9, A. (Imi; 20 mg / kg), aposinine (Apo; 15 mg / kg), or vitamin C (Vit C; 15 mg / kg) for 1 hour before the onset of cortical stress (2 h, 3-14 days) ) Was administered intraperitoneally.

As a result, when physical stress (2-h detention) was repeatedly applied for 3, 7, or 14 days, ROS increased according to the number of repetitions (MDA analysis). However, as a result of intraperitoneal administration of aposinin (15 mg / kg) daily before stress, the lipid peroxidation phenomenon was completely blocked in the brain of the subject mouse, and the hippocampus of imipramine (20 mg / kg) Was also inhibited by lipid peroxidation. Whereas vitamin C (15 mg / kg) partially inhibited ROS production (Fig. 9, B).

The behavior observation results are shown in Figs. 9, C and D. Fig. In stressed mice, immobility time increased in TST and FST, but this phenomenon disappeared by the administration of aposinine (15 mg / kg) and imipramine (20 mg / kg). In this case, vitamin C (15 mg / kg) was not effective. These results demonstrate that aposinin not only inhibits stress-induced increases in ROS but also has anti-depressant effects in animal models. In addition, it was found that administration of a substance capable of inhibiting the activity or expression of p47phox or p67phox can prevent the depression from being formed due to repeated stress. Data are presented as mean ± SEM (n = 8-10), with * and ** representing significant differences between the two groups, p <0.05 and p <0.01, respectively. One-way ANOVA and Newman-Keuls multiple comparison tests were used.

Although aposinin relieves depression and anxiety due to chronic stress, its neuroanatomical target site is not known precisely. Thus, expression of p47phox in specific brain regions was specifically blocked using shRNA using the lentiviral vector system. For this, a non-viral vector control, Lenti shRNA-GFP control, and Lenti-p47phox-shRNA test group were used. The Lenti-GFP control or Lenti-p47phox-shRNA infiltrated independently into the dentate area.

The results are shown in FIG. A shows the expression of GFP in HeLa and A549 cells at 8 hours after infection with Lenti-p47phox-shRNA expressing GFP. B is a graphical representation of the period of stress and time of test using experimental design, P47phox-shRNA (GFP expression) or Lenti-GFP in tooth marrow (DG) of the hippocampus of both sides of the section. After 15 days of challenge, mice were subjected to 2h-14d prick stress, and behavior was observed 15 days later. The Lenti-shRNA-GFP control group showed typical depression-like behavior in TST and FST compared to the control group in which the non-viral vector was delivered, but the Lenti-47phox-shRNA test group did not show depressive behavior (Fig. 10, ). These results indicate that the suppression of p47phox expression in specific areas of the brain alone can treat and prevent stress-related depressive behaviors. Data were expressed as mean ± SEM (n = 5-6), with * and ** representing p <0.05 and p <0.01, respectively, showing significant differences from the 24-hour control. One-way ANOVA and Newman-Keuls multiple comparison tests were used.

Similar experiments were performed using p47phox genetic mouse (p47phox +/-). The genotypes of mice with two copies (+ / +) of the p47phox gene and only one copy of the genetically modified mouse were analyzed by PCR and then analyzed by MspI restriction enzyme cleavage. The results are shown in FIG. Mice with the above genotype were subjected to 2h-14d stress followed by TST and FST behavioral evaluation. The experimental results are shown in Fig. 11, C (TST) and D (FST). In TST and FST, non - transgenic mice exhibited depressive - like behavior, but this was not observed in p47phox +/- mice. Data are presented as mean ± SEM (n = 12-15) and ** is significant at p <0.01 compared to control mice. One-way ANOVA and Newman-Keuls multiple comparison tests were used. These results all support that the deficit of p47phox has an antidepressant effect.

The experimental results of this embodiment are summarized as follows. Treatment of apopticin with chronic stressed animal models showed antistress and antidepressant effects similar to those of imipramine, a treatment for depression. In addition, microinjection of Lenti-p47phox-shRNA into the hippocampus did not produce depressive behavior due to chronic stress, unlike the control group, when NADPH oxidase activity was inhibited. It was also confirmed that depressive behavior was not produced by chronic stress in p47phox mutant (+/-) mice, unlike the wild (+ / +) control group. The increase of NADPH oxidase expression as a candidate gene for oxidative stress in the chronic stress animal model was confirmed, and it was confirmed that SH-SY5Y human neuronal cell line was treated with glucocorticoid, which is a stress hormone, In the stress model, NADPH oxidase expression was increased, oxidative stress was increased, and mitochondrial function was impaired, and NADPH oxidase suppression and oxidative stress were blocked by inhibiting new protein synthesis. In addition, the expression of NADPH oxidase, p47phox and p67phox was also increased in blood cells of chronic stressed animals as well as brain.

In addition, when SH-SY5Y human nerve cell line was treated with glucocorticoid, apocyanin inhibited cell membrane movement (inhibition of enzyme activation) of p47phox, a key subunit of NADPH oxidase. It was confirmed that increase of intracellular oxidative stress was suppressed by siRNA against p47phox gene under glucocorticoid treatment condition. Accordingly, the present invention relates to a method of treating depression, anxiety, fear memory, mental panic disorder, obsessive compulsive disorder, post-traumatic stress disorder, schizophrenia, or degenerative disorder, which is caused by chronic stress that targets NADPH oxidase, A therapeutic agent for brain diseases (Alzheimer's Dementia, Parkinson's disease, stroke, ALS, etc.), or a drug capable of preventing the disease at the stage before the onset of a mental disorder in which the symptoms are triggered or deteriorated by chronic stress .

<110> Ewha University - Industry Collaboration Foundation <120> Therapeutic agents targeting p47phox or p67phox for stress          disorder and method of screening thereof <130> PA110519KR <150> KR10-2011-0004369 <151> 2011-01-17 <160> 44 <170> Kopatentin 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for genomic DNA from mouse <400> 1 tggaagaagc tgagagttga gg 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for genomic DNA from mouse <400> 2 tccaggagct tatgaatgac c 21 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 3 agaaggtggt gaagcaggca tc 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 4 cgaaggtgga agagtgggag ttg 23 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for beta-actin <400> 5 atcgtgcgtg acatcaaaga gaag 24 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for beta-actin <400> 6 tgccacagga ttccataccc aag 23 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for p47phox <400> 7 accggctatt tcccatcc 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for p47phox <400> 8 tggatgctct gtgcgttg 18 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for p67phox <400> 9 catgaagcac accatccagt cc 22 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for p67phox <400> 10 gatttctctt cctgtttgtc catctg 26 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for COX-2 <400> 11 gcttcaaaca gtttctctac aacaa 25 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for COX-2 <400> 12 catttcttcc cccagcaac 19 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for NOS <400> 13 tgtggctacc acattgaaga a 21 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for NOS <400> 14 tcatgataac gtttctggct ctt 23 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for NF-kappa b <400> 15 ttgtctgaca tgggtctcca 20 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for NF-kappa b <400> 16 gcactgtctt ctttcacctc tgt 23 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TNF-alpha <400> 17 tgcctatgtc tcagcctctt c 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TNF-alpha <400> 18 gaggccattt gggaacttct 20 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for IL-1 beta <400> 19 tgagcacctt cttttccttc a 21 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for IL-1 beta <400> 20 ttgtctaatg ggaacgtcac ac 22 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for SOD1 <400> 21 tgcgtgctga agggcgac 18 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for SOD1 <400> 22 gtcctgacaa cacaactggt tc 22 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for CAT <400> 23 tctacacaaa ggtgttgaac gag 23 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for CAT <400> 24 agggtggacg tcagtgaaat 20 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for HO-1 <400> 25 ctgctagcct ggtgcaaga 19 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for HO-1 <400> 26 ccaacaggaa gctgagagtg a 21 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PRX1 <400> 27 tggattctca cttctgtcat ctgg 24 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PRX1 <400> 28 gcgcttggga tctgatatta aggg 24 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for human p47phox <400> 29 cacggacaac cagacaaa 18 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for human p47phox <400> 30 tcttctcgta gttggcaatg 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for human p67phox <400> 31 cccctttcag aagacagcat 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for human p67phox <400> 32 tcatctggaa agccttggtc 20 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for gp91-phox <400> 33 ggtttatgat gatgggccta a 21 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for gp91-phox <400> 34 gcactggaac ccctgagaaa 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for p22-phox <400> 35 ccccggggaa agaggaaaaa 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for p22-phox <400> 36 aggacagccc ggacgtagta 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for p67-phox <400> 37 gggaaccagc tgatagacta 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for p67-phox <400> 38 tccattcctc yttcttggca 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for p47-phox <400> 39 cagccagcac tatgtgtaca 20 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for p47-phox <400> 40 gaactcgtag atctcggtga a 21 <210> 41 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> forward primer for p40-phox <400> 41 tggcccagca gctgcg 16 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for p40-phox <400> 42 cgrtagcggc ggtagatgag 20 <210> 43 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense strand for siRNA-p47phox <400> 43 cuauuuccca uccauguau 19 <210> 44 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> antisense strand for siRNA-p47phox <400> 44 auacauggau gggaaauag 19

Claims (20)

delete delete delete delete a depression-improving food or feed containing a siRNA of p47phox mRNA consisting of a sense strand represented by the sequence of SEQ ID NO: 43 and an antisense strand represented by the sequence of SEQ ID NO: 44 inhibiting the expression or activity of p47phox or Lenti-p47phox-shRNA . delete delete p47phox-shRNA as an active ingredient, the siRNA of p47phox mRNA consisting of a sense strand represented by the sequence of SEQ ID NO: 43 inhibiting the expression or activity of p47phox and an antisense strand represented by the sequence of SEQ ID NO: 44, Or &lt; / RTI &gt; delete delete A sense strand represented by the sequence of SEQ ID NO: 43, and an antisense strand represented by the sequence of SEQ ID NO: 44, as an active ingredient. delete the siRNA or the Lenti-p47phox-shRNA of p47phox mRNA composed of the sense strand represented by the sequence of SEQ ID NO: 43 inhibiting the expression or activity of p47phox and the antisense strand represented by the sequence of SEQ ID NO: 44, A method of preventing or treating depression. (1) a first step of providing isolated cells expressing p47phox or p67phox;
(2) a second step of bringing the test substance into contact with the cell; And
(3) a third step of selecting a candidate substance as a candidate substance in which at least one of the expression, activity and function of the p47phox or p67phox is inhibited in cells contacted with the test substance as compared with the cell not in contact with the test substance &Lt; / RTI &gt; delete 15. The screening method according to claim 14, wherein the expression, activity, or inhibition of the function of p47phox or p67phox is determined by measuring ROS (Reactive Oxygen Species) production. 15. The screening method according to claim 14, wherein the cells of the first step are provided in the form of an experimental animal. delete Measuring the expression, activity and function of p47phox or p67phox of a biological sample separated from the subject. The information providing method according to claim 19, wherein the biological sample is blood, and the diagnosis is for predicting the onset of depression or the prognosis of depression.

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