A composition for removing intracellular reactive oxygen species containing CPNE7 protein or a peptide derived from CPNE7 protein

Abstract

The present invention relates to a composition for removing intracellular reactive oxygen species containing CPNE7 protein or a functional peptide (Selcopintide) derived from CPNE7 protein, a polynucleotide encoding the peptide, an expression vector containing the polynucleotide, and a pharmaceutical composition, a quasi-drug composition, and a health functional food composition containing the peptide.

Description

Technical Field

[0001] The present invention relates to a composition for removing reactive oxygen species, and more particularly, to a composition for removing intracellular reactive oxygen species containing CPNE7 protein or a peptide derived from CPNE7 protein.

Background Art

[0002] The development of medical technology has extended the average lifespan of humans through disease prevention and treatment. Recently, with the growth of the elderly population, interest in the prevention and treatment of various geriatric diseases has been increasing. However, the exact mechanism that causes aging has not yet been elucidated, and technologies related to the causes and treatment of numerous geriatric diseases remain in the initial stage of development. According to the "2021 Elderly Statistics" of Statistics Korea, the population aged 65 and over accounts for 16.5% of the total, and it is predicted that this proportion will increase annually. An aging society defined by the United Nations is a society in which the population aged 65 and over accounts for more than 7% of the total population. Based on this, Korea has already entered an aging society, and with the continuous increase in the average lifespan, the proportion of the elderly population is expected to further increase.

[0003] As the elderly population increases, the importance of developing anti-aging technologies that enable the prevention and treatment of age-related diseases is becoming increasingly significant. Regarding anti-aging technologies, in order to understand aging at the individual level, it is necessary to first investigate the aging phenomenon from a cell-molecular biological perspective. According to the free radical theory (Hartman et al., 1986), which is the most widely accepted theory regarding the cause of aging, reactive oxygen species (ROS) with strong reactivity cause somatic mutations and damage proteins, which are regarded as the cause of aging. ROS are structurally very unstable and can bind to DNA within cells, causing strong oxidation. Highly toxic oxygen molecule radicals bind to unsaturated fatty acids (PUFAs) in cell membranes, resulting in changes in cell membranes and ultimately promoting cell aging. Numerous diseases such as tissue damage caused by ischemia / reperfusion, metabolic syndrome, Parkinson's disease, neurodegenerative diseases such as Alzheimer's, progeria, cancer, and cardiovascular diseases are not only related to ROS, but ROS also promote skin aging by deforming the forms of collagen and elastin, which mainly exist in the dermis layer of the skin.

[0004] Endogenous antioxidant enzymes (such as SOD, Catalase, Glutathione peroxidase, etc.) naturally produced in the human body function to neutralize reactive oxygen species by stably changing their structure. However, it has been reported that this decreases after the age of 30, and it is known to decrease by about 50% at the age of 40 compared to 25 years old, and up to 90% by the age of 60s. Therefore, the production rate of reactive oxygen species increases as aging progresses. Secondary antioxidants, which are non-enzymatic exogenous antioxidants (such as vitamin C, E, A, selenium, etc.), are quickly absorbed after ingestion and thus cannot sufficiently compensate for the decrease in endogenous antioxidant enzymes.

[0005] In copine-7 (CPNE7) protein-deficient mice, it was experimentally confirmed that the aging of odontoblasts that form dentin is accelerated and pathological dentin is formed. CPNE7 is a protein that induces the regeneration of dentin, which constitutes more than about 70% of the tooth structure. Focusing on the fact that the CPNE7 protein is an essential element for maintaining homeostasis in odontoblasts, the research team considered that the deficiency of CPNE7 leads to DNA damage by causing excessive formation of reactive oxygen species due to a decrease in endogenous antioxidant enzymes in odontoblasts, leading to the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a composition for removing intracellular reactive oxygen species containing a CPNE7 protein or a peptide derived from the CPNE7 protein.

[0007] Another object of the present invention is to provide a polynucleotide encoding the peptide.

[0008] Another object of the present invention is to provide an expression vector containing the polynucleotide.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, containing the peptide.

[0010] Another object of the present invention is to provide a quasi-drug composition for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, containing the peptide.

[0011] Another object of the present invention is to provide a health functional food composition for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, containing the peptide.

[0012] The object of the present invention is not limited to those mentioned above, and other objects not mentioned will be clearly understood by those having ordinary knowledge in the technical field to which the present invention pertains from the following description.

Means for Solving the Problems

[0013] As a result of conducting various studies to solve the above technical problems, the present inventors have developed a composition for removing intracellular reactive oxygen species, which contains CPNE7 protein or a peptide derived from CPNE7 protein that removes intracellular reactive oxygen species.

[0014] In addition, a novel peptide consisting of 10 amino acids for a preparation that can treat heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, which is derived from CPNE7 protein, has been developed.

[0015] As one embodiment for achieving the above object, the present invention provides a peptide for treating heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, which contains the amino acid sequence of the following general formula 1. K - Y - R1 - R2 - R3 - R4 - R5 - R6 - R7 - R8 (General formula 1) In the above general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q), R2 is arginine (R) or glutamine (Q), R3, R4 and R5 are each arginine (R) or lysine (K), R6 is asparagine (N) or serine (S), R7 and R8 are lysine (K) or tyrosine (Y).

[0016] The peptide provided by the present invention shows no cytotoxicity and can increase the expression levels of the Dspp, Dmp1, and Nestin genes, which are odontoblast differentiation marker genes. When transplanted into the living body together with dental pulp tissue cells, the dental pulp tissue cells may exhibit the characteristic of forming dentin / dental pulp tissue-like tissue.

[0017] As long as the peptide provided by the present invention can show a therapeutic effect on heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, variant peptides having a sequence in which one or more amino acid residues are different from the amino acid sequence constituting it are also included in the scope of the peptides provided by the present invention.

[0018] Generally, amino acid exchanges in proteins and polypeptides that do not globally change the activity of the molecule are known in the art. The most commonly occurring exchanges are exchanges between the amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In addition, it may include peptides in which the structural stability of the peptide against heat, pH, etc. is increased or the ability to scavenge intracellular reactive oxygen species is increased due to mutations or modifications in the amino acid sequence.

[0019] For example, glutamine, an acidic amino acid located at the 3rd position of the peptide of SEQ ID NO: 1 provided by the present invention, can still exhibit the same effect as the peptide provided by the present invention even if it is substituted with lysine or arginine, which are basic amino acids; arginine, a basic amino acid located at the 4th or 5th position of the peptide of SEQ ID NO: 1, can still exhibit the same effect as the peptide provided by the present invention even if it is substituted with glutamine, an acidic amino acid, or lysine, a basic amino acid; lysine, a basic amino acid located at the 6th, 7th, or 9th position of the peptide of SEQ ID NO: 1, can still exhibit the same effect as the peptide provided by the present invention even if it is substituted with arginine, a basic amino acid, or tyrosine, an aromatic amino acid; asparagine, an acidic amino acid located at the 8th position of the peptide of SEQ ID NO: 1, can still exhibit the same effect as the peptide provided by the present invention even if it is substituted with serine, a neutral amino acid; tyrosine, an aromatic amino acid located at the 10th position of the peptide of SEQ ID NO: 1, can still exhibit the same effect as the peptide provided by the present invention even if it is substituted with lysine, a basic amino acid.

[0020] Thus, the acidic amino acids, basic amino acids, or aromatic amino acids that make up the peptide of the present application can each still exhibit the same effect as the peptide provided by the present invention even if they are substituted with different acidic amino acids, basic amino acids, neutral amino acids, or aromatic amino acids. Therefore, it is obvious that mutant peptides having an amino acid sequence different from that of the peptide of the present invention by one or more amino acid residues are also included in the scope of the peptide provided by the present invention.

[0021] In addition, even if the peptide of the present invention has a form in which any amino acid is added to its N-terminus or C-terminus, it can still exhibit the effects of the peptide provided by the present invention as it is, and thus is included in the scope of the peptide provided by the present invention. As an example, it may be in a form in which 1 to 300 amino acids are added to the N-terminus or C-terminus of the peptide. As another example, it may be in a form in which 1 to 100 amino acids are added to the N-terminus or C-terminus of the peptide. As still another example, it may be in a form in which 1 to 24 amino acids are added to the N-terminus or C-terminus of the peptide.

[0022] In another aspect, the present invention provides a polynucleotide encoding the peptide.

[0023] The polynucleotide can be mutated by substitution, deletion, insertion, or a combination thereof of one or more bases. When chemically synthesizing and manufacturing the nucleotide sequence, synthetic methods known in the art, for example, the methods described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), can be used, and it can be synthesized using phosphotriester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoligation methods, oligonucleotide synthesis methods on solid supports, etc. For example, the polynucleotide encoding the peptide of the present invention may contain the nucleotide sequence of SEQ ID NO: 4.

[0024] In still another aspect, the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for producing the peptide using the transformant.

[0025] As used herein, the term "expression vector" of the present invention refers to a recombinant vector capable of expressing a target peptide in a target host cell, and means a genetic construct containing essential regulatory elements operably linked so that the gene insert is expressed. The expression vector contains expression regulatory elements such as a start codon, a stop codon, a promoter, and an operator. The start codon and the stop codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and must always function in an individual when the genetic construct is administered, and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

[0026] As used herein, the term "operably linked" means a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked so as to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA are operably linked and can affect the expression of the coding sequence. The operative linkage with the expression vector can be produced using genetic recombination techniques known in the art, and site-specific DNA cleavage and ligation can use enzymes generally known in the art.

[0027] Furthermore, the expression vector may contain a signal sequence for the excretion of the peptide in order to facilitate the separation of the peptide from the cell culture medium. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, it is necessary to provide an exogenous translation regulatory signal that may include the ATG start codon. These exogenous translation regulatory signals and start codons can be from various natural and synthetic sources. The expression efficiency can be increased by the introduction of appropriate transcription or translation enhancing factors.

[0028] In addition, the expression vector may further include a protein tag that can be optionally removed using an endopeptidase to facilitate detection of the peptide.

[0029] The term "tag" in the present invention means a molecule that exhibits a quantifiable activity or property, and may be a fluorescent molecule including a chemical fluorescent substance such as fluorescein, or a polypeptide fluorescent substance such as green fluorescent protein (GFP) or a related protein, and may also be an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, or a streptavidin tag. In particular, when using an epitope tag, a peptide tag preferably composed of 6 or more amino acid residues, more preferably composed of 8 to 50 amino acid residues can be used.

[0030] The term "coronary artery disease (CAD)" in the present invention means a disease in which the coronary artery, which is a major blood vessel of the heart, becomes narrow or blocked. As a result, oxygen and nutrients may become insufficient in the heart muscle, and such a phenomenon mainly occurs due to arteriosclerosis. Arteriosclerosis occurs when cholesterol, fat, calcium, etc. accumulate on the blood vessel wall, and oxidative stress plays a major role in this process. Oxidative stress is a disease caused by oxidative stress of cardiomyocytes that worsens blood vessel damage, causes inflammation, and accelerates arteriosclerosis.

[0031] The term "myocardial infarction (MI)" in the present invention may mean a situation in which the blood supply to the coronary artery is suddenly blocked and the heart muscle stops. Oxidative stress can worsen damage to cardiomyocytes, promote an inflammatory response, and induce a decline in heart function.

[0032] The term "heart failure" in the present invention means a state in which the heart cannot supply sufficient blood to the body. Oxidative stress in cardiomyocytes in such a situation can, as one of the main causes of heart failure, worsen cell damage and inflammation and reduce heart function.

[0033] The term "atrial fibrillation" in the present invention may mean a state in which abnormal electrical signals occur in the atria of the heart, causing the heart rhythm to become fast and irregular. As a result, the heart cannot efficiently supply blood to the body. Oxidative stress in cardiomyocytes leads to an increase in oxygen-reactive compounds (free radicals) in an unbalanced state within the cells, and such oxidative compounds can damage cells and biomolecules, thus worsening the electrical imbalance in the heart and increasing the likelihood of atrial fibrillation.

[0034] Oxidative stress in cardiomyocytes may exacerbate heart damage and inflammation, which can impair the electrical stability of the heart and cause heart disease. Therefore, reducing oxidative stress in cardiomyocytes may enable the treatment of heart disease.

[0035] The term "Photodermatitis" in the present invention means a state in which ultraviolet rays or a combination of specific substances and sunlight cause an inflammatory reaction in the skin. One of the main causes of skin damage due to ultraviolet exposure is oxidative stress. Oxidative stress can affect dermal fibroblasts, thereby promoting collagen degradation and inflammatory reactions. Such processes can change the properties of the skin and exacerbate the symptoms of photodermatitis.

[0036] The term "Atopic dermatitis" of the present invention refers to a chronic dermatitis characterized by dry skin, accompanied by itching symptoms, and repeated inflammation. It is known that genetic factors, environmental factors, immune system abnormalities, etc. are related, and the stress of dermal fibroblasts may be associated with the onset and progression of atopic dermatitis. Oxidative stress generates free radicals that damage cells, which causes an inflammatory response and a decrease in cell function. Since dermal fibroblasts are located in the dermal layer of the skin and play an important role in maintaining the structure and function of the skin, when the function of dermal fibroblasts decreases due to oxidative stress, the protective function of the skin may be weakened. As a result, the skin becomes more vulnerable to external stimuli, and the symptoms of atopic dermatitis may worsen. The oxidative stress caused by this can promote the inflammatory response. Such an inflammatory response is one of the main symptoms of atopic dermatitis and is the cause of the inability of dermal fibroblasts to function properly. Furthermore, cell damage caused by oxidative stress can exacerbate the damage to the skin barrier. When the skin barrier is damaged, substances that cause allergic reactions are more likely to penetrate the skin, which can further worsen the symptoms of atopic dermatitis.

[0037] In the present invention, the expression vector may contain a nucleotide sequence encoding a CPNE7 protein or a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. However, the vector used in this case is not particularly limited as long as it can produce the peptide. Preferably, it is plasmid DNA, phage DNA, etc. More preferably, it can be a commercially developed plasmid (such as pUC18, pBAD, pIDTSAMRT-AMP), a plasmid derived from Escherichia coli (such as pYG601BR322, pBR325, pUC118, pUC119), a plasmid derived from Bacillus subtilis (such as pUB110, pTP5), a plasmid derived from yeast (such as YEp13, YEp24, YCp50), phage DNA (such as Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP), an animal virus vector (such as retrovirus, adenovirus, vaccinia virus), an insect virus vector (such as baculovirus). Since the expression level and modification of proteins may vary depending on the host cell, it is desirable to select and use the host cell that is most suitable for the purpose.

[0038] The transformant provided by the present invention is prepared by introducing the expression vector provided by the present invention into a host for transformation, and can be used to express the polynucleotide contained in the expression vector to produce the peptide. The transformation can be carried out by various methods, and as long as the peptide can be produced, it is not particularly limited thereto. Examples include the CaCl2 precipitation method, the Hanahan method in which the efficiency is increased by using a reducing substance called DMSO (dimethyl sulfoxide) in the CaCl2 precipitation method, the electroporation method, the calcium phosphate precipitation method, the protoplast fusion method, the stirring method using silicon carbide fibers, the Agrobacterium-mediated transformation method, the transformation method using PEG, the dextran sulphate, lipofectamine, and the transformation method mediated by drying / suppression. Also, the host used for the preparation of the transformant is not particularly limited as long as it can produce the peptide, and examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; fungal cells such as Pichia pastoris; insect cells such as Drosophila and Spodoptera frugiperda Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; or plant cells.

[0039] The transformant can also be used in a method for producing a peptide consisting of the amino acids of SEQ ID NO: 1 of the present invention. Specifically, the method for producing a peptide consisting of the amino acids of SEQ ID NO: 1 of the present invention may include (a) culturing the transformant to obtain a culture, and (b) recovering the peptide of the present invention from the culture.

[0040] The term "cultivation" in the present invention means a method of growing microorganisms under artificially adjusted environmental conditions as appropriate. In the present invention, the method of cultivating the transformant can be carried out by a method known in the art. Specifically, the cultivation is not particularly limited as long as it can produce by expressing the peptide consisting of the amino acid consisting of SEQ ID NO: 1 of the present invention, and can be continuously cultivated by a batch process, a fed-batch culture or a repeated fed-batch process.

[0041] The medium used for cultivation needs to meet the requirements of a specific strain by an appropriate method while adjusting temperature, pH, etc. under aerobic conditions in a normal medium containing an appropriate carbon source, nitrogen source, amino acids, vitamins, etc. As the carbon source that can be used, a mixed sugar of glucose and xylose is used as the main carbon source, and in addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil; fatty acids such as palmitic acid, stearic acid, linoleic acid; alcohols such as glycerol, ethanol; and organic acids such as acetic acid are included. These substances can be used alone or as a mixture. Also, as the nitrogen source that can be used, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products can be used. These nitrogen sources can be used alone or in combination. The medium may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts as a phosphorus source. As the phosphorus source that can be used, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salt is included. Also, as inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate can be used. Finally, in addition to the above substances, substances essential for growth such as amino acids and vitamins can be used.

[0042] In addition, appropriate precursors can be used in the culture medium. The above-mentioned raw materials can be added to the culture by batch, fed-batch or continuous methods by appropriate methods during the culture process, but are not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acidic compounds such as phosphoric acid or sulfuric acid can be used by appropriate methods to adjust the pH of the culture.

[0043] Also, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress bubble generation. To maintain an aerobic state, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 35°C. The culture is continued until the maximum amount of the peptide is obtained. For that purpose, it is usually achieved in 10 hours to 100 hours.

[0044] Furthermore, the step of recovering the peptide from the culture can be performed by methods known in the art. Specifically, the recovery method is not particularly limited thereto as long as it can be used for the recovery of the produced peptide, but preferably, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used.

[0045] The pharmaceutical composition of the present invention further comprises, in addition to the peptide, a suitable carrier (natural or non-natural carrier), excipient or diluent commonly used in the preparation of pharmaceutical compositions, and can be prepared in the form of a pharmaceutical composition for treating heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts. Specifically, the pharmaceutical composition can be formulated and used in the form of a sterile injectable solution that can be administered to the site where a heart disease or a skin disease is induced by a conventional method. In the present invention, examples of the carrier, excipient and diluent that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, collagen and the like. When formulating, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are commonly used. In particular, it may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried product, a suppository, an ointment (for example, a pulp liner, etc.). Examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of the base for suppositories include witepsol (registered trademark), macrogol (registered trademark), tween (registered trademark) 61, cacao butter, laurin fat, glycerogelatin and the like.

[0046] The content of the peptide contained in the pharmaceutical composition of the present invention is not particularly limited, but can be contained in an amount of 0.0001 to 50% by weight, more preferably 0.01 to 20% by weight, based on the total weight of the final composition.

[0047] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" in the present invention means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dosage level can be determined based on the severity of the disease, the activity of the drug, the age, weight, health status, gender of the patient, the sensitivity of the patient to the drug, the administration time, administration route, and excretion ratio of the composition of the present invention used, the treatment period, factors including drugs formulated or used concomitantly with the composition of the present invention, and other factors known in the medical field. The pharmaceutical composition of the present invention can be administered alone or in combination with a pharmaceutical composition for treating heart diseases caused by oxidative stress of cardiomyocytes or skin diseases caused by oxidative stress of dermal fibroblasts, which are known. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects.

[0048] The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art considering the purpose of use, the severity of the disease, the age, weight, gender, medical history of the patient, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng / kg to about 100 mg / kg, preferably 1 ng / kg to about 10 mg / kg per adult. The administration frequency of the composition of the present invention is not particularly limited thereto, but it can be administered once a day or the dose can be divided and administered several times a day. The above dosage does not limit the scope of the present invention in any way.

[0049] In another aspect, the present invention provides a method for treating heart diseases caused by oxidative stress of cardiomyocytes or skin diseases caused by oxidative stress of dermal fibroblasts, which includes the step of administering the pharmaceutical composition in a pharmaceutically effective amount to an individual other than a human being in whom a heart disease caused by oxidative stress of cardiomyocytes or a skin disease caused by oxidative stress of dermal fibroblasts has developed.

[0050] As used herein, the term "individual" may include, without limitation, mammals such as rats and livestock for which treatment of heart disease caused by oxidative stress of cardiomyocytes or skin disease caused by oxidative stress of dermal fibroblasts is required, excluding humans from among those individuals in whom the disease has developed.

[0051] The administration route of the pharmaceutical composition for treating heart disease caused by oxidative stress of cardiomyocytes or skin disease caused by oxidative stress of dermal fibroblasts of the present invention can be administered via any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention can be administered via routes such as oral administration, cutaneous administration, intramuscular injection, intravenous injection, subcutaneous injection, respiratory administration, rectal administration, and local administration, although not particularly limited thereto, depending on the purpose.

[0052] "Oral administration (intraoral administration)" is a method of ingesting a drug through the mouth, and generally tablets, capsules, or drugs in liquid form can be used. "Cutaneous administration" is a method of applying or affixing a drug to the skin, and can be used in forms such as creams, gels, and patches. "Intramuscular injection" is a method of injecting a drug directly into muscle tissue, and mainly a syringe and needle can be used. "Intravenous injection" is a method of injecting a drug directly into a blood vessel, and a syringe and needle or an intravenous cannula can be used. "Subcutaneous injection" is a method of injecting a drug into the adipose tissue under the skin and can be used for drug administration such as insulin. "Respiratory administration" is a method of delivering a drug to the respiratory tract through the mouth or nose and can be used in forms such as inhalers, gases, and vapors. "Rectal administration" is a method of injecting a drug into the rectum and can be used in forms such as rectal drugs and rectal drops. "Local administration" is a method of directly applying a drug to the site that requires treatment and can be used in forms such as creams, gels, and patches depending on the characteristics of the disease.

[0053] In another aspect, the present invention provides a quasi-drug composition for preventing or improving heart disease caused by oxidative stress of cardiomyocytes or skin disease caused by oxidative stress of dermal fibroblasts, containing the peptide.

[0054] As used herein, the term "improvement" means all acts that at least reduce a parameter related to the condition to be treated, such as the degree of symptoms.

[0055] In the present invention, the above-mentioned "improvement" means administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual in need of treatment for a heart disease caused by oxidative stress of cardiomyocytes or a skin disease caused by oxidative stress of dermal fibroblasts, and promoting the removal of reactive oxygen species in cardiomyocytes or dermal fibroblasts, so that the symptoms of a heart disease caused by oxidative stress of cardiomyocytes or a skin disease caused by oxidative stress of dermal fibroblasts are improved or become beneficial. It can be interpreted as meaning all acts.

[0056] As used herein, the term "quasi-drug" means an article that is used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans and animals and has a milder action than pharmaceuticals. For example, according to the Pharmaceutical Affairs Law, quasi-drugs exclude articles used for the uses of pharmaceuticals, and include fiber and rubber products used for the treatment and prevention of human and animal diseases, articles that have a mild action on the human body or do not act directly, articles similar to those that are not instruments or machines, and bactericides and insecticides for preventing infectious diseases.

[0057] In the present invention, the types and dosage forms of the quasi-drug composition containing the peptide are not particularly limited. As an example, it can be an ointment, a patch, a powder, a gelling agent, a tablet, or a spray.

[0058] In another aspect, the present invention provides a health functional food composition for preventing or improving a heart disease caused by oxidative stress of cardiomyocytes or a skin disease caused by oxidative stress of dermal fibroblasts, which contains the peptide.

[0059] In the terms of the present invention, the term "food" includes meat, sausage, bread, chocolate, candies, snacks, confectioneries, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, drink agents, alcoholic beverages, vitamin complexes, functional foods and health foods, etc., and includes all foods in the ordinary sense.

[0060] The above-mentioned "functional food" is the same term as "food for special health use (FoSHU)", and means a food with high medical and therapeutic effects that is processed so that its physiological regulatory function can be efficiently manifested in addition to nutrient supply. Note that "functional" means to regulate nutrients or obtain useful effects for health purposes such as physiological effects on the structure and function of the human body. The food of the present invention can be manufactured by methods commonly used in the industry, and during the above-mentioned manufacturing, raw materials and components commonly added in the industry can be added for manufacturing. Also, the dosage form of the above-mentioned food can be manufactured without limitation as long as it is a dosage form recognized as food. The food composition of the present invention is manufactured in various dosage forms. Different from general pharmaceuticals, it has the advantage of using food as a raw material and having no side effects that may occur during long-term use of pharmaceuticals. It is excellent in portability. The food of the present invention can be ingested as an adjuvant for enhancing the preventive or ameliorative effect on heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts.

[0061] The above-mentioned "health food" means a food that has a positive health maintenance and promotion effect compared to general foods, and "health supplement food" means a food for health supplement purposes. In some cases, the terms "functional food", "health food", and "health supplement food" can be used interchangeably.

[0062] Specifically, the health functional food is a food obtained by adding the peptide of the present invention to food materials such as beverages, teas, spices, gums, and confectioneries, or manufacturing it as a capsule, powder, suspension, etc. Intaking this means bringing about specific health effects. Different from general pharmaceuticals, it has the advantage of having no side effects that may occur during long-term intake of pharmaceuticals when using food as the raw material.

[0063] Since the food composition of the present invention can be taken daily, it can be expected to have a high effect on the prevention or improvement of heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, and thus can be used very usefully.

[0064] The food composition may further contain a physiologically acceptable carrier, but the type of the carrier is not particularly limited, and any carrier commonly used in the technical field can be used.

[0065] In addition, the food composition may contain additional components that are commonly used in food compositions and can improve odor, taste, visual appearance, etc. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. It may also contain amino acids such as lysine, tryptophan, cysteine, valine, etc.

[0066] Furthermore, the food composition may contain food additives such as preservatives (e.g., potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate), bactericides (e.g., bleaching powder and high - grade bleaching powder, sodium hypochlorite), antioxidants (e.g., butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT)), colorants (e.g., tar dyes), color developers (e.g., sodium nitrite), bleaching agents (e.g., sodium sulfite), seasonings (e.g., sodium glutamate (MSG)), artificial sweeteners (e.g., dulcin, cyclamate, saccharin, sodium), flavors (e.g., vanilla, lactones), swelling agents (e.g., alum (potassium aluminum sulfate), potassium hydrogen D - tartrate), fortifiers, emulsifiers, thickeners (pastes), coating agents, gum bases, defoamers, solvents, improvers. The additives can be selected according to the type of food and used in appropriate amounts.

[0067] The peptide of the present invention can be added as it is or used together with other foods or food ingredients and can be appropriately used by ordinary methods. The mixing amount of the active ingredient can be suitably determined according to its purpose of use (prevention, health or therapeutic treatment). Generally, when manufacturing foods or beverages, the food composition of the present invention can be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, based on 100 parts by weight of the food or beverage composition. However, when ingested for a long time for health and hygiene purposes, it may contain a content below the above - mentioned range, and since there is no problem from the perspective of safety, the active ingredient can also be used in an amount above the above - mentioned range.

[0068] An example of the food composition of the present invention may be used as a healthy beverage composition. In that case, like ordinary beverages, various flavoring agents or natural carbohydrates etc. may be included as additional ingredients. The aforementioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetening agent may use natural sweeteners such as thaumatin and stevia extract; synthetic sweeteners such as saccharin and aspartame. The ratio of the aforementioned natural carbohydrates may usually be about 0.01 g to 0.04 g, specifically about 0.02 g to 0.03 g per 100 mL of the healthy beverage composition of the present invention.

[0069] In addition to the above, the healthy beverage composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents etc. In addition, it may contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. Such components may be used independently or in combination. The ratio of such additives is not very important, but it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the healthy beverage composition of the present invention.

[0070] If the food composition of the present invention can show a preventive or ameliorating effect on heart diseases due to oxidative stress of cardiomyocytes or skin diseases due to oxidative stress of dermal fibroblasts, it may be included in various weight percentages. Specifically, the peptide of the present invention may be included in the food composition in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight based on the total weight of the food composition, but is not limited thereto.

Advantages of the Invention

[0071] The present invention can provide a composition for removing intracellular reactive oxygen species containing CPNE7 protein or a peptide derived from CPNE7 protein.

[0072] Another object of the present invention may be to provide a polynucleotide encoding the peptide.

[0073] Another object of the present invention may be to provide an expression vector containing the polynucleotide.

[0074] Another object of the present invention may be to provide a pharmaceutical composition for preventing or treating heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, which contains the peptide.

[0075] Another object of the present invention may be to provide a quasi-drug composition for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, which contains the peptide.

[0076] Another object of the present invention may be to provide a health functional food composition for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, which contains the peptide.

[0077] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those with ordinary knowledge in the technical field to which the present invention belongs from the following description.

Brief Description of the Drawings

[0078]

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Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Mode for Carrying Out the Invention

[0079] The object and effect of the present invention, and the technical configuration for achieving them, will become clear by referring to the examples described in detail below together with the accompanying drawings. In the description of the present invention, when it is determined that a specific description of a known function or configuration may obscure the gist of the present invention, the detailed description thereof will be omitted. And the terms described below are terms defined for the description of the examples in the present invention, and these may vary depending on the intention or convention of the user, operator, etc.

[0080] However, the present invention is not limited to the examples disclosed below and can be realized in various different forms. These examples are provided only to make the disclosure of the present invention complete and to inform those with ordinary knowledge in the technical field to which the present invention belongs of the category of the invention, and the present invention is defined only by the scope of the claims. Therefore, the definition should be made based on the content throughout this specification. Hereinafter, the examples of the present invention will be specifically described.

[0081] (Example 1: Comparison of Reactive Oxygen Species Levels Using the Cell-Permeable Reagent 2’,7’-Dichlorofluorescein Diacetate (DCFDA)) Human dental pulp cells (hDPCs) were cultured in α-MEM medium containing 10% FBS under conditions of 5% CO2 and 37°C.

[0082] Next, the cultured hDPCs cells were seeded at 4×10 per 60 mm culture dish 5After dispensing by cell count and culturing for 24 hours, the cultured cells were transformed by introducing the Shcon vector and the ShCpne7 vector using Lipofectamine Plus (registered trademark) reagent.

[0083] The transformed hDPCs cells were again dispensed into a 96-well plate at 3×10 3 cells per well, cultured for 24 hours, then treated with the respective DCFDA substances, and after 40 minutes, the reactive oxygen species level was measured at Ex / Em = 485 / 535 nm using a microplate reader. At this time, as a control group, a control group that was not treated with anything and a group using the Shcon vector were used.

[0084] Referring to Figure 1, it can be confirmed that when Cpne7 decreased due to ShCpne7 transformation, the reactive oxygen species level increased by about 2 times or more compared to the control group. From this, it can be seen that Cpne7 is effective in maintaining the reactive oxygen species level at a normal level in hDPCs cells.

[0085] (Example 2. Comparison of reactive oxygen species levels using the cell-permeable reagent 2’,7’-dichlorofluorescein diacetate (DCFDA)) The DCFDA substances were respectively treated on sections of mandibular molar tissues of 6-month-old mice in which the Cpne7 gene was knocked out, and after 40 minutes, the reactive oxygen species levels were respectively measured using a confocal microscope. At this time, the mandibular molars of 6-month-old BL6 mice were used as a control group.

[0086] When the Cpne7 gene was removed, it was confirmed that the reactive oxygen species level in odontoblasts in vivo increased compared to the control group, similar to the in vitro results (Figure 2).

[0087] From the above results, it was confirmed that Cpne7 is a factor that maintains the reactive oxygen species level at a normal level in vivo including odontoblasts.

[0088] (Example 3. Verification of oxidative stress vulnerability upon Cpne7 knockdown using odontoblasts) The cultured hDPCs cells were dispensed into a 96-well plate at a cell count of 3×10 per well, cultured for 24 hours, and then treated with 50 μM and 100 μM of hydrogen peroxide (H2O2) for 24 hours. After adding the WST-8 solution to each well and reacting for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader. 3

[0089] As a result of the measurement, in the group in which the Cpne7 gene was knocked down (ShCpne7), the cell viability was significantly decreased compared to the control group at a concentration of 100 μM of H2O2 (Figure 3). Therefore, it was confirmed that Cpne7 is related to maintaining the reactive oxygen species level at a normal level in hDPCs cells.

[0090] (Example 4. Comparative confirmation of DNA damage degree by immunofluorescence staining method of γ-H2A.X, a DNA damage marker protein) From the results of Figures 1 and 2, it was confirmed that Cpne7 is involved in maintaining the normal level of reactive oxygen species, and in particular, reactive oxygen species increase when Cpne7 is knocked out. Thus, it was confirmed whether the increase in reactive oxygen species can increase DNA damage in odontoblasts.

[0091] Immunofluorescence staining was performed on the mandibular molar tissue sections of 6-month-old Cpne7 knockout mice using a γ-H2A.X antibody, and the intensities were compared. At this time, mandibular molar tissue sections of 6-month-old BL6 mice were used as the control group.

[0092] As can be confirmed from Figure 4, it was confirmed that DNA damage has accumulated in the odontoblasts of Cpne7 knockout mice in which reactive oxygen species have increased.

[0093] Thus, it was analyzed that the increase in reactive oxygen species due to Cpne7 knockout induces DNA damage.

[0094] (Example 5. Comparison of cell senescence degree by staining of B-gal, a cell senescence marker enzyme) ​From the results of FIGS. 1, 2, and 4, it was confirmed that when Cpne7 was knocked out, increased reactive oxygen species induced DNA damage, and it was confirmed whether the induced DNA damage could promote cell senescence.

[0095] The transformed hDPCs were dispensed into 35-mm culture dishes at a cell number of 1×10 5 and cultured for 24 hours. After that, cell differentiation was carried out until day 21 while replacing the α-MEM medium containing 5% FBS, 50 μg / ml ascorbic acid, and 10 mM β-glycerophosphate every two days. The differentiated odontoblasts on days 0, 4, 7, 14, and 21 were fixed with 4% paraformaldehyde, and B-gal staining was performed in a 37°C incubator without CO2 for 24 hours.

[0096] In addition, B-gal staining was performed for 24 hours on tissue sections of the mandibular molars of 6-month-old Cpne7 knockout mice in which reactive oxygen species and DNA damage were confirmed. As shown in FIG. 5, it was confirmed that there were more B-gal positive cells in the odontoblasts in which the Cpne7 gene was knocked down or knocked out than in the control group.

[0097] Therefore, it was confirmed that the increase in reactive oxygen species due to Cpne7 knockout induced DNA damage and promoted cell senescence.

[0098] (Example 6. Histomorphological comparison of mandibular molars of 6-month-old and 12-month-old Cpne7 knockout mice) H&E (Hematoxylin and Eosin) staining was performed on tissue sections of the mandibular molars of 6-month-old and 12-month-old Cpne7 knockout mice.

[0099] From FIGS. 1, 2, 3, and 4 above, it was confirmed that Cpne7 knockout odontoblasts promoted DNA damage induction and cell senescence due to an increase in reactive oxygen species.

[0100] As a result of the histological comparison of the mandibular molars of 6-month-old and 12-month-old Cpne7 knockout mice, it was confirmed that the odontoblasts of the mandibular molars of 6-month-old Cpne7 knockout mice showed an irregular arrangement and the cell nuclei showed abnormal morphology. In addition, it was confirmed that pathological bone-like hard tissue was formed in the pulp tissue.

[0101] In the dental tissues of 12-month-old Cpne7 knockout mice, more pathological bone-like hard tissue was formed in the pulp tissue. It was confirmed that most of the odontoblasts had died and the remaining odontoblasts were less healthy compared to the control group.

[0102] Therefore, it was confirmed that the increase in reactive oxygen species due to Cpne7 knockout induces DNA damage and cellular senescence in odontoblasts, causing a pathological condition.

[0103] (Example 7. After treating with H2O2 that induces intracellular reactive oxygen species and treating with CPNE7 recombinant protein, cell confirmation was performed under an optical microscope) hDPCs were dispensed into a 35 mm culture dish at a cell number of 1×10 5 After culturing for 24 hours, H2O2 was treated at concentrations of 5 μM, 100 μM, 1 mM, or 2 mM, respectively, and then 100 ng / ml of recombinant CPNE7 protein was treated, followed by culturing for 24 hours. After the culture was completed, the cultured cells were washed with PBS and observed under an optical microscope. At this time, as a control group, hDPCs cultured without treating with the recombinant CPNE7 protein (rCPNE7) were used.

[0104] As can be confirmed from Figure 7, no changes were observed at concentrations of H2O2 of 100 μM or less, and cell death due to oxidative stress was observed at concentrations of 1 mM or more. On the other hand, in the recombinant CPNE7 protein treatment group, a higher cell survival rate was observed compared to the control group. Thus, it was analyzed that the CPNE7 protein has the efficacy of removing reactive oxygen species.

[0105] (Example 8. Comparison of Reactive Oxygen Species Levels Using the Cell-Permeable Reagent 2’,7’-Dichlorofluorescein Diacetate (DCFDA)) Cultured hDPCs were dispensed into a 35-mm culture dish for confocal microscopy at a density of 1×10 5 cells, cultured for 24 hours, treated with 1 mM H2O2, treated with 100 ng / ml rCPNE7, and photographed with a confocal microscope for approximately 1 hour.

[0106] Referring to Fig. 8, after treatment with 1 mM H2O2, the amount of reactive oxygen species reached its maximum value at approximately 30 minutes and then decreased, and significance was confirmed at approximately 1 hour. This confirmed the possibility of CPNE7 protein removing reactive oxygen species.

[0107] (Example 9. Comparison of DNA Damage Levels by Immunofluorescence Staining of the DNA Damage Marker Protein γ-H2A.X) From Fig. 8 above, it was confirmed that the CPNE7 recombinant protein removes excess reactive oxygen species in hDPCs, and based on this, the degree of DNA damage was confirmed.

[0108] Cultured hDPCs were dispensed into a 35-mm culture dish for confocal microscopy at a density of 1×10 5 cells, cultured for 24 hours, treated with 1 mM H2O2, treated with 100 ng / ml rCPNE7, and fixed with 4% paraformaldehyde after 24 hours.

[0109] Immunofluorescence staining was performed on the fixed cells using a γ-H2A.X antibody, and the intensities were compared.

[0110] Referring to Fig. 9, as a result of the comparison, a significant amount of DNA damage was induced in the group treated with 1 mM H2O2 to impose oxidative stress, whereas in the group treated with 100 ng / ml of the CPNE7 recombinant protein, a significant decrease in the accumulation of DNA damage was confirmed. This confirmed that the CPNE7 protein can prevent or repair DNA damage by removing excess reactive oxygen species.

[0111] (Example 10. Comparison of cell senescence levels by staining with B-gal, a cell senescence marker enzyme) In FIGS. 8 and 9, it has been confirmed that the CPNE7 recombinant protein prevents or repairs DNA damage by removing excessive reactive oxygen species in hDPCs, and thus, the effect of this on cell senescence was to be confirmed.

[0112] Therefore, cultured hDPCs were dispensed into 6-well plates at a cell number of 1×10 5 and cultured for 24 hours. Then, 1 mM of H2O2 was treated, and 100 ng / ml of rCPNE7 was treated. After 12 hours or 24 hours, the cells were fixed with 4% paraformaldehyde respectively.

[0113] Next, the fixed cells were subjected to B-gal staining for 24 hours to compare the degree of cell senescence.

[0114] Referring to FIG. 10, after treatment with 1 mM of H2O2 for 12 hours, B-gal positive hDPCs cells were confirmed, and more were confirmed after 24 hours. On the other hand, in the CPNE7 recombinant protein treatment group, it was confirmed that the B-gal positive hDPCs cells were significantly decreased compared to the H2O2 alone treatment group. Thereby, it was confirmed that the reactive oxygen species scavenging ability of the CPNE7 protein can suppress cell senescence by preventing or repairing DNA damage.

[0115] (Example 11. Measurement of viable cell mass using WST-8) Cultured hDPCs cells were dispensed into 96-well plates at a cell number of 3×10 per well 3 and cultured for 24 hours. Then, hydrogen peroxide (H2O2) was treated at 0 μM (Control), 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, and 2 mM respectively for 24 hours. After adding the WST-8 solution to each well and reacting for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader.

[0116] As can be confirmed from Fig. 11, it was confirmed that the cell viability decreased with the increase in the H2O2 concentration, and it was confirmed that there was almost no change in the cell viability at a concentration of 500 uM or more of H2O2. Thereafter, a concentration of 1 mM of H2O2 was selected as the oxidative stress-imparting concentration.

[0117] (Example 12. Synthesis of Peptide for Removing Intracellular Reactive Oxygen Species) The inventors of the present invention synthesized a peptide (SEQ ID NO: 1) showing an intracellular reactive oxygen species scavenging effect using 9-fluorenylmethyloxycarbonyl (Fmoc), and substituted the amino acids of the synthesized peptide to synthesize peptides for each group (Tables 1 to 12). N-KYQRRKKNKY-C (SEQ ID NO: 1) First, the peptides of Group 1 were synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 or the peptide of SEQ ID NO: 1 with lysine or arginine (Table 1). [Table 1]

[0118] Next, the peptides of Group 2 were synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine and substituting the 8th amino acid with serine (Table 2).

[0119] [Table 2]

[0120] Next, the peptides of Group 3 were synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine and substituting the 9th amino acid with tyrosine (Table 3).

[0121] [Table 3]

[0122] Next, the peptides of Group 4 were synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine, substituting the 8th amino acid with serine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 4).

[0123] [Table 4]

[0124] Next, the peptides of Group 5 were synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO: 1 with arginine, substituting the 4th amino acid with glutamine, and substituting the 5th to 7th amino acids with lysine or arginine (Table 5).

[0125] [Table 5]

[0126] Next, the peptides of Group 6 were synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO: 1 with arginine, substituting the 4th amino acid with glutamine, substituting the 5th to 7th amino acids with lysine or arginine, and substituting the 8th amino acid with serine (Table 6).

[0127] [Table 6]

[0128] Next, the peptides of Group 7 were synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO: 1 with arginine, substituting the 4th amino acid with glutamine, substituting the 5th to 7th amino acids with lysine or arginine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 7).

[0129] [Table 7]

[0130] Next, the peptide of Group 8 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO: 1 with arginine, the 4th amino acid with glutamine, the 5th to 7th amino acids with lysine or arginine, the 8th amino acid with serine, the 9th amino acid with tyrosine, and the 10th amino acid with lysine (Table 8).

[0131] [Table 8]

[0132] Next, the peptide of Group 9 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO: 1 with lysine, the 4th amino acid with glutamine, and the 5th to 7th amino acids with lysine or arginine (Table 9).

[0133] [Table 9]

[0134] Next, the peptide of Group 10 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO: 1 with lysine, the 4th amino acid with glutamine, the 5th to 7th amino acids with lysine or arginine, and the 8th amino acid with serine (Table 10).

[0135] [Table 10]

[0136] Next, the peptide of Group 11 was synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO: 1 with lysine, the 4th amino acid with glutamine, the 5th to 7th amino acids with lysine or arginine, the 9th amino acid with tyrosine, and the 10th amino acid with lysine (Table 11).

[0137] [Table 11]

[0138] Finally, the peptide of Group 12 was synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 12).

[0139]

Table 12

[0140] (Example 13. Confirmation of the Ability to Scavenge Reactive Oxygen Species by Concentration of the Functional Peptide (Selcopintide) Derived from CPNE7 Protein) Figure 12 shows the results of cell confirmation under an optical microscope and measurement of the amount of viable cells using WST-8 after treating with H2O2 that induces intracellular reactive oxygen species and then treating with Selcopintide (SEQ ID NO: 96).

[0141] The cultured hDPCs cells were dispensed into 6-well plates at a cell number of 1×10 5 per well, cultured for 24 hours, then treated with 1 mM hydrogen peroxide (H2O2), and then treated with Selcopintide at concentrations of 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml for 24 hours. As a control group, hDPCs that were not treated with anything were used.

[0142] In the group treated with 1 mM H2O2, significant cell death of odontoblasts was confirmed compared to the control group. On the other hand, in the Selcopintide-treated group, it was confirmed that the cell viability was higher than that in the group treated with H2O2 alone. In particular, in the 1 μg / ml and 10 μg / ml treatment groups, it was confirmed that the cell viability was high (Figure 12).

[0143] The cultured hDPCs cells were dispensed into 96-well plates at 3×10 3After dispensing by cell count and culturing for 24 hours, hydrogen peroxide (H2O2) was treated at 1 mM, and then Selcopintide was treated at concentrations of 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml for 24 hours. As a control group, hDPCs that were not treated with anything were used.

[0144] After adding the WST-8 solution to each well and reacting for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader. It has been confirmed that the cell viability was significantly decreased in the H2O2-treated group compared to the control group, but it was confirmed that the cell viability was significantly higher in the 1 μg / ml and 10 μg / ml treatment groups of Selcopintide compared to the H2O2-alone treatment group (Figure 12).

[0145] Thereby, the reactive oxygen species scavenging ability of the CPNE7 protein-derived functional peptide (Selcopintide) was confirmed.

[0146] (Example 14. Confirmation of the reactive oxygen species scavenging ability of the CPNE7 protein-derived functional peptide (Selcopintide) according to the concentration by comparing the reactive oxygen species levels using the cell-permeable reagent 2′,7′-dichlorofluorescein diacetate (DCFDA)) The cultured hDPCs were dispensed into a 35-mm confocal microscope culture dish at a cell count of 1×10 5 After culturing for 24 hours, 1 mM of H2O2 was treated, and 10 μg / ml of Selcopintide was treated, and photographed with a confocal microscope for about 1 hour.

[0147] Referring to Figure 13, after treatment with 1 mM of H2O2, the amount of reactive oxygen species showed a maximum value at about 30 minutes, and then decreased, and significance was confirmed at about 1 hour. Thereby, the possibility of Selcopintide scavenging reactive oxygen species was confirmed.

[0148] (Example 15. Confirmation of the mRNA level of the endogenous antioxidant enzyme of Selcopintide in hDPCs cells by RT-PCR) The cultured hDPCs were placed in a 60-mm culture dish at 4×105 Dispense to obtain the cell count, culture for 24 hours, then treat with 1 mM H2O2, and after treating with CPNE7 recombinant protein (100 ng / ml) and Selcopintide (10 μg / ml), the mRNA levels of Sod1, 2, 3, Catalase, Gpx7, Hmox1, and Txrnd1 were confirmed 30 minutes and 1 hour later.

[0149] Referring to Figure 14, it can be confirmed that 30 minutes after the treatment with CPNE7 recombinant protein and Selcopintide, the endogenous antioxidant enzymes Sod1, 2, and Catalase were significantly increased compared to the group treated with H2O2 alone. After 1 hour of treatment with CPNE7 recombinant protein and Selcopintide, it can be confirmed that the endogenous antioxidant enzymes Sod1, Gpx7, and Hmox1 and Txrnd1, which inhibit the formation of OH free radicals, were significantly increased.

[0150] Thus, it was confirmed that Selcopintide can remove reactive oxygen species by regulating endogenous antioxidant enzymes in the same manner as CPNE7 protein.

[0151] (Example 16. Comparative confirmation of DNA damage degree by immunofluorescence staining of γ-H2A.X, a DNA damage marker protein) As described above, it was confirmed that the CPNE7 recombinant protein suppresses cell aging by preventing or repairing DNA damage by removing excessive reactive oxygen species in hDPCs.

[0152] The cultured hDPCs were dispensed into a 35 mm culture dish for confocal microscopy at 1×10 5 Dispense to obtain the cell count, culture for 24 hours, then treat with 1 mM H2O2, and after treating with 10 μg / ml Selcopintide, the cells were fixed with 4% paraformaldehyde 24 hours later.

[0153] Immunofluorescence staining was performed on the fixed cells using a γ-H2A.X antibody, and the intensities were compared.

[0154] Referring to Fig. 15, in the group treated with 1 mM of H2O2 to impose oxidative stress, a significant amount of DNA damage was induced. In contrast, in the group treated with 10 μg / ml of Selcopintide, it was confirmed that the accumulation of DNA damage was significantly reduced.

[0155] Thereby, it was confirmed that Selcopintide prevents or repairs DNA damage by removing excessive reactive oxygen species, similar to the CPNE7 protein.

[0156] (Example 17. Measurement of the amount of viable cells using WST-8) In the aforementioned odontoblast research, it was confirmed that the CPNE7 protein and Selcopintide have the ability to remove reactive oxygen species. Subsequently, similar to odontoblasts, the ability of the peptide to remove reactive oxygen species was also confirmed in cardiomyocytes, which are known to differentiate in mesenchymal stem cells (MSCs).

[0157] Cultured cardiomyocyte (H9C2 cell line) cells were dispensed into a 96-well plate at a cell number of 3×10 3 per well and cultured for 24 hours. Then, hydrogen peroxide (H2O2) was treated at 0 μM (control group), 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, and 2 mM for 24 hours. After adding the WST-8 solution to each well and reacting for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader.

[0158] As shown in Fig. 16, it was confirmed that the cell viability decreased with the increase in the H2O2 concentration, and it was confirmed that there was almost no change in the cell viability at a concentration of H2O2 of 500 μM or more.

[0159] Thereafter, a concentration of 1 mM of H2O2 was selected as the oxidative stress-imposing concentration in H9C2 cells.

[0160] (Example 17. After treating with H2O2 that induces intracellular reactive oxygen species and then treating with Selcopintide, cell confirmation was performed under an optical microscope and the amount of viable cells was measured using WST-8) The cultured H9C2 cells were dispensed into 6-well plates at a cell count of 1×10 5 per well, cultured for 24 hours, then treated with 1 mM hydrogen peroxide (H2O2), and then treated with Selcopintide at concentrations of 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml for 24 hours. As a control group, untreated H9C2 cells were used.

[0161] In the group treated with 1 mM H2O2, significant cell death of cardiomyocytes was confirmed compared to the control group. On the other hand, in the Selcopintide-treated group, it was confirmed that the cell survival rate was higher compared to the group treated with H2O2 alone (Figure 17).

[0162] The cultured H9C2 cells were dispensed into 96-well plates at a cell count of 3×10 3 per well, cultured for 24 hours, then treated with 1 mM hydrogen peroxide (H2O2), and then treated with Selcopintide at concentrations of 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml for 24 hours. As a control group, untreated H9C2 cells were used.

[0163] After adding the WST-8 solution to each well and reacting for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader.

[0164] It has been confirmed that the cell survival rate decreased significantly in the H2O2-treated group compared to the control group. However, compared to the group treated with H2O2 alone, it was confirmed that the cell survival rate was significantly higher in the groups treated with 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml of Selcopintide (Figure 17).

[0165] Thus, it was confirmed that the functional peptide (Selcopintide) derived from the CPNE7 protein also has the ability to remove reactive oxygen species in cardiomyocytes.

[0166] (Example 18. Comparison of cell senescence levels by staining with B-gal, a cell senescence marker enzyme) From FIG. 17, it has been confirmed that Selcopintide is effective in scavenging excessive reactive oxygen species in H9C2 cells, and thus, the effect of this on cell senescence was to be confirmed.

[0167] The cultured H9C2 cells were dispensed into a 6-well plate at a cell number of 1×10 5 , cultured for 24 hours, then treated with 1 mM H2O2, and treated with 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml of Selcopintide. After 24 hours, the cells were fixed with 4% paraformaldehyde respectively.

[0168] Next, the fixed cells were stained with B-gal for 24 hours to compare the degree of cell senescence.

[0169] After 24 hours of treatment with 1 mM H2O2, a large number of B-gal positive H9C2 cells were confirmed. On the other hand, in the Selcopintide treatment group, it was confirmed that the number of B-gal positive H9C2 cells was significantly decreased compared to the group treated with H2O2 alone.

[0170] This was analyzed to indicate that the reactive oxygen species scavenging ability of Selcopintide can suppress the senescence of cardiomyocytes.

[0171] (Example 19. Measurement of viable cell mass using WST-8) In the studies on the odontoblasts and cardiomyocytes mentioned above, it was confirmed that Selcopintide has the ability to scavenge reactive oxygen species. Next, similar to the odontoblasts and cardiomyocytes, it was decided to confirm whether the peptide has the ability to scavenge reactive oxygen species in dermal fibroblasts, which are known to differentiate in MSCs.

[0172] The cultured dermal fibroblasts (CCD986SK) were dispensed into a 96-well plate at 3×10 per well 3After dispensing by cell count and culturing for 24 hours, hydrogen peroxide (H2O2) was treated at 0 uM (control group), 50 uM, 100 uM, 200 uM, 500 uM, 1 mM, and 2 mM for 24 hours each. After adding the WST-8 solution to each well and reacting for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader.

[0173] As shown in Fig. 19, it was confirmed that the cell viability decreased as the H2O2 concentration increased, and it was confirmed that there was almost no change in cell viability at a concentration of 500 uM or more of H2O2.

[0174] Thereafter, a concentration of 1 mM of H2O2 was selected as the oxidative stress-inducing concentration in CCD986SK cells.

[0175] (Example 20. After treating with H2O2 that induces intracellular reactive oxygen species and then treating with Selcopintide, cell confirmation was performed under an optical microscope and the amount of viable cells was measured using WST-8.) The cultured CCD986SK cells were dispensed into a 6-well plate at 1×10 5 cells per well, cultured for 24 hours, treated with hydrogen peroxide (H2O2) at 1 mM, and then treated with Selcopintide at concentrations of 1 ug / ml, 10 ug / ml, 100 ug / ml, and 500 ug / ml for 24 hours. As a control group, CCD986SK cells that were not treated with anything were used.

[0176] In the 1 mM H2O2 treatment group, significant cell death of dermal fibroblasts was confirmed compared to the control group. On the other hand, in the Selcopintide treatment group, it was confirmed that the cell viability was higher than that in the H2O2 alone treatment group, and particularly, the significance was the highest at 1 ug / ml and 10 ug / ml of Selcopintide (Fig. 20).

[0177] The cultured CCD986SK cells were placed in a 96-well plate at 3×10 3After dispensing by cell count and culturing for 24 hours, hydrogen peroxide (H2O2) was treated at 1 mM, and then Selcopintide was treated at concentrations of 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml for 24 hours. As a control group, CCD986SK cells that were not treated with anything were used.

[0178] After adding the WST-8 solution to each well and reacting for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader.

[0179] It has been confirmed that the cell viability was significantly decreased in the H2O2-treated group compared to the control group. However, it was confirmed that the cell viability was significantly higher in the Selcopintide 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml treatment groups compared to the H2O2-alone treatment group (Figure 20).

[0180] Therefore, it was confirmed that the functional peptide (Selcopintide) derived from the CPNE7 protein also has the ability to scavenge reactive oxygen species in dermal fibroblasts.

[0181] (Example 21. Comparison of cell senescence degree by staining of B-gal, a cell senescence marker enzyme) As confirmed in Figure 20 above, it has been confirmed that Selcopintide is effective in scavenging excessive reactive oxygen species in CCD986SK cells, and the effect of this on cell senescence was to be confirmed.

[0182] The cultured CCD986SK cells were dispensed into a 6-well plate at a cell number of 1×10 5 After culturing for 24 hours, 1 mM of H2O2 was treated, and 1 μg / ml, 10 μg / ml, 100 μg / ml, and 500 μg / ml of Selcopintide were treated, and the cells were fixed with 4% paraformaldehyde after 24 hours.

[0183] The fixed cells were stained with B-gal for 24 hours to compare the degree of cell senescence.

[0184] After 24 hours of treatment with 1 mM H2O2, a large number of B-gal positive CCD986SK cells were confirmed. On the other hand, in the Selcopintide treatment group, it was confirmed that the number of B-gal positive CCD986SK cells was significantly reduced compared to the group treated with H2O2 alone.

[0185] This was analyzed to indicate that the reactive oxygen species scavenging ability of Selcopintide can suppress the aging of dermal fibroblasts.

[0186] From the foregoing examples, it can be seen that the CPNE7 protein and the functional peptide (Selcopintide) derived from the CPNE7 protein can be used to prevent or treat pulp diseases, myocardial infarction, skin inflammation, etc. caused by excessive reactive oxygen species, thereby preventing the aging of an individual.

[0187] This specification and the drawings disclose preferred embodiments of the present invention. Although specific terms are used, these are merely used in a general sense to clearly explain the technical content of the present invention and assist in understanding the invention, and are not intended to limit the scope of the present invention. It is obvious to those with ordinary knowledge in the technical field to which the present invention pertains that other modifications based on the technical idea of the present invention are possible in addition to the embodiments disclosed herein.

Claims

1. A composition for removing intracellular reactive oxygen species, comprising a CPNE7 protein or a peptide consisting of the amino acids of any of the sequences of SEQ ID NOs: 1 to 96.

2. The composition for removing intracellular reactive oxygen species according to claim 1, wherein the cells are dental pulp cells, cardiomyocytes or dermal fibroblasts.

3. A peptide consisting of the amino acids of any of the sequences of SEQ ID NOs: 1 to 96 for removing intracellular reactive oxygen species.

4. A polynucleotide encoding the peptide according to claim 3.

5. An expression vector comprising the polynucleotide according to claim 4.

6. A pharmaceutical composition for preventing or treating heart diseases caused by oxidative stress of cardiomyocytes or skin diseases caused by oxidative stress of dermal fibroblasts, comprising the peptide according to claim 3.

7. The composition according to claim 6, wherein the pharmaceutical composition comprises a polypeptide in which the peptide is repeatedly linked.

8. The composition according to claim 6, wherein the pharmaceutical composition comprises a complex in which a drug showing a preventive or therapeutic effect on heart diseases caused by oxidative stress of cardiomyocytes or skin diseases caused by oxidative stress of dermal fibroblasts is bound to the peptide.

9. The composition according to claim 6, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

10. The composition according to claim 6, wherein the heart disease is coronary artery disease (CAD), myocardial infarction (MI), heart failure or atrial fibrillation (AF).

11. The composition according to claim 6, wherein the skin disease is photodermatitis or atopic dermatitis.

12. A quasi-drug composition for preventing or improving heart diseases caused by oxidative stress of cardiomyocytes or skin diseases caused by oxidative stress of dermal fibroblasts, comprising the peptide according to claim 3.

13. The composition according to claim 12, wherein the quasi-drug composition is provided as an ointment, patch, powder, gelling agent, tablet or spray.

14. A health functional food composition for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, which contains the peptide according to claim 3.

15. The composition according to claim 14, wherein the health functional food composition is used for the production of beverages, teas, spices, gums, or confectioneries.

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