Composition for removing intracellular reactive oxygen species comprising CPNE7 protein or peptide derived from CPNE7 protein

Abstract

The present invention relates to a composition for the removal of reactive oxygen species in cells, said cells comprising a CPNE7 protein or a functional peptide derived from a CPNE7 protein, the invention also relates to a polynucleotide encoding the peptide, an expression vector containing the polynucleotide, and a pharmaceutical composition, a foreign medicine composition and a health-care functional food composition containing the peptide.

Description

Technical Field

[0001] The present invention relates to a composition for removing active oxygen, and more particularly, to a composition for removing active oxygen in cells containing a CPNE7 protein or a peptide derived from a CPNE7 protein. Background Art

[0002] The development of medical technology has extended the average life span of humans by preventing and treating diseases. Recently, with the growth of the elderly population, attention has been paid to the prevention and treatment of various geriatric diseases. However, the exact mechanism of aging has not yet been clarified, and the causes and treatment technologies of many geriatric diseases are still in the early stages of development. According to the "2021 Senior Population Statistics" of the National Statistical Office, the population over 65 years old accounts for 16.5% of the total population, and the proportion is expected to increase year by year. The United Nations defines an aging society as a society where the population over 65 years old accounts for more than 7% of the total population. Based on this, South Korea is already in the state of entering an aging society, and as the average life expectancy continues to increase, the proportion of the elderly population is expected to increase further.

[0003] With the increase in the elderly population, the development of anti-aging technologies that can prevent and treat senile diseases is becoming increasingly important. In order to understand aging at the individual level, it is necessary to first study the aging phenomenon based on the perspective of cell-molecular biology in order to understand anti-aging technologies. In the research on the causes of aging, according to the reactive oxygen theory (free radical theory, Hartman et al., 1986), which is recognized as the most favorable theory, highly reactive reactive oxygen species (ROS) cause somatic mutations and protein damage, which are shown as the cause of aging. Reactive oxygen species are extremely unstable in structure and can bind to DNA in cells to cause strong oxidation. The highly toxic oxygen molecule free radicals (radicals) cause changes in the cell membrane by binding to the unsaturated fatty acids (PUFA) of the cell membrane, ultimately promoting cell aging. Reactive oxygen species are not only associated with numerous diseases such as tissue damage caused by ischemia / reperfusion, metabolic syndrome, degenerative neurological diseases such as Parkinson's disease and Alzheimer's disease, premature aging, cancer, cardiovascular disease, etc., but also can deform the morphology of collagen and elastin, which are mainly present in the dermis of the skin, thereby promoting skin aging.

[0004] Endogenous antioxidant enzymes (SOD, catalase, glutathione peroxidase, etc.) naturally produced in the human body can stabilize the structure of active oxygen and have the function of neutralizing active oxygen. However, it is reported that this function decreases with age over 30 years old, and decreases by about 50% at the age of 40 compared to 25 years old, and decreases by 90% at the age of 60 years old. Therefore, the generation rate of active oxygen increases with aging. Secondary antioxidants such as non-enzymatic exogenous antioxidants (vitamin C, E, A, selenium, etc.) can be absorbed quickly after ingestion, and cannot fully compensate for the reduction of endogenous antioxidant enzymes.

[0005] Experiments have confirmed that in mice lacking the Copine-7 (CPNE7) protein, the aging of odontoblasts, which form dentin, is accelerated, forming pathological dentin. CPNE7 is a protein that induces the regeneration of more than about 70% of the dentin that constitutes the tooth structure. The research team focused on the fact that CPNE7 protein is an essential element for maintaining homeostasis in odontoblasts, and believed that the loss of CPNE7 would cause excessive formation of reactive oxygen species due to the reduction of endogenous antioxidant enzymes in odontoblasts, which in turn led to DNA damage, and thus completed the present invention. Summary of the invention

[0006] Technical issues

[0007] An object of the present invention is to provide a composition for removing active oxygen in cells containing a CPNE7 protein or a peptide derived from a CPNE7 protein.

[0008] Another object of the present invention is to provide a polynucleotide encoding the peptide.

[0009] Another object of the present invention is to provide an expression vector comprising the polynucleotide.

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

[0011] Another object of the present invention is to provide a quasi-drug composition comprising the peptide for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts.

[0012] Another object of the present invention is to provide a health functional food composition comprising the peptide for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts.

[0013] The objects of the present invention are not limited to the above-mentioned contents, and a person skilled in the art can clearly understand other objects not mentioned from the following description.

[0014] Solution to the problem

[0015] In order to solve the above technical problems, the present inventors have developed a composition for removing active oxygen in cells comprising CPNE7 protein or a peptide derived from CPNE7 protein after various studies, which can remove active oxygen in cells.

[0016] Furthermore, a novel peptide consisting of 10 amino acids for use in a pharmaceutical preparation capable of treating heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts derived from CPNE7 protein was developed.

[0017] As one embodiment for achieving the above object, the present invention provides a peptide comprising an amino acid sequence of the following general formula 1, for treating heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts.

[0018] KY-R1-R2-R3-R4-R5-R6-R7-R8 (Formula 1)

[0019] In the general formula 1,

[0020] R1 is arginine (R), lysine (K) or glutamine (Q);

[0021] R2 is arginine (R) or glutamine (Q);

[0022] R3, R4 and R5 are arginine (R) or lysine (K), respectively;

[0023] R6 is asparagine (N) or serine (S); and

[0024] R7 and R8 are lysine (K) or tyrosine (Y).

[0025] The peptide provided in the present invention has no cytotoxicity and can increase the expression levels of Dspp, Dmp1 and Nestin genes, which are marker genes for odontoblast differentiation. When transplanted into the body together with dental pulp tissue cells, the dental pulp tissue cells can show the characteristics of forming dentin / pulp tissue-like tissue.

[0026] The peptides provided in the present invention, as long as they can show therapeutic effects on heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, even mutant peptides with one or more amino acid residues different from the amino acid sequence constituting them are included in the scope of the peptides provided in the present invention.

[0027] Generally, the exchange of amino acids in proteins and polypeptides without changing the activity of the molecule as a whole is well known in the art to which the present invention belongs. The most common exchange is the exchange between amino acid residues and 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, the peptides may include peptides whose heat, pH, etc. related structural stability or intracellular active oxygen removal ability is increased by mutation or modification in the amino acid sequence.

[0028] For example, even if glutamine, which is an acidic amino acid at position 3 of the peptide of sequence number 1 provided in the present invention, is replaced by lysine or arginine, which is a basic amino acid, the effect of the peptide provided in the present invention can be directly expressed; even if arginine, which is a basic amino acid at position 4 or 5 of the peptide of sequence number 1, is replaced by glutamine, which is an acidic amino acid, or lysine, which is a basic amino acid, the effect of the peptide provided in the present invention can be directly expressed; even if lysine, which is a basic amino acid at position 6, 7 or 9 of the peptide of sequence number 1, is replaced by arginine, which is a basic amino acid, or tyrosine, which is an aromatic amino acid, the effect of the peptide provided in the present invention can be directly expressed; even if asparagine, which is an acidic amino acid at position 8 of the peptide of sequence number 1, is replaced by serine, which is a neutral amino acid, the effect of the peptide provided in the present invention can be directly expressed; even if tyrosine, which is an aromatic amino acid at position 10 of the peptide of sequence number 1, is replaced by lysine, which is a basic amino acid, the effect of the peptide provided in the present invention can be directly expressed.

[0029] As described above, the acidic amino acids, basic amino acids or aromatic amino acids constituting the peptide of the present invention can directly exhibit the effects of the peptide provided in the present invention even if they are replaced by different acidic amino acids, basic amino acids, neutral amino acids or aromatic amino acids. Therefore, it is obvious that mutant peptides having a sequence different from that of one or more amino acid residues constituting the amino acid sequence of the peptide of the present invention also fall within the scope of the peptides provided in the present invention.

[0030] Furthermore, 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, the effect of the peptide provided in the present invention can be directly expressed, and therefore it is included in the category of the peptide provided in the present invention. As one example, it can be 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 can be a form in which 1 to 100 amino acids are added to the N-terminus or C-terminus of the peptide, and as yet another example, it can be a form in which 1 to 24 amino acids are added to the N-terminus or C-terminus of the peptide.

[0031] As another embodiment, the present invention provides a polynucleotide encoding the peptide.

[0032] For the polynucleotide, more than one base can be mutated by substitution, knockout, insertion or a combination thereof. In the case of preparing by chemically synthesizing a nucleotide sequence, a synthesis method known in the art to which the present invention belongs can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem Int Ed Engl., 37: 73-127, 1988), and can be synthesized using triester, phosphite, phosphoramidite and H-phosphate methods, polymerase chain reaction (PCR) and other automatic primer methods, oligonucleotide synthesis on a solid support, etc. For example, the polynucleotide encoding the peptide of the present invention may include the base sequence of sequence number 4.

[0033] As another embodiment, the present invention provides an expression vector comprising the polynucleotide, a transformant comprising the expression vector, and a method for preparing the peptide using the transformant.

[0034] The term "expression vector" of the present invention refers to a recombinant vector that can express a target peptide in a target host cell, and means a gene construct that contains necessary regulatory elements that are operably linked to express a gene insert. The expression vector contains expression regulatory elements such as a start codon, a stop codon, a promoter, and an operator gene. The start codon and the stop codon are usually considered to be part of the nucleotide sequence encoding the polypeptide, and must exhibit an effect in an individual when the gene construct is administered, and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

[0035] The term "operably linked" of the present invention refers to a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked in order to perform a common function. For example, operably linking a promoter and a nucleic acid sequence encoding a protein or RNA can affect the expression of the coding sequence. Operable linkage with an expression vector can be prepared using gene recombination techniques known in the art to which the present invention belongs, and site-specific DNA cleavage and ligation can use enzymes generally known in the art to which the present invention belongs.

[0036] In addition, in order to promote the separation of peptides from the cell culture fluid, the expression vector may include a signal sequence for discharging the peptides. Specific initiation signals may also be required when performing effective translation of the inserted nucleotide sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, an exogenous translation regulation signal that may include the ATG initiation codon may be provided. These exogenous translation regulation signals and initiation codons may be a variety of natural and synthetic sources. Expression efficiency may be increased by introducing appropriate transcription or translation enhancers.

[0037] Meanwhile, in order to facilitate the detection of the peptide, the expression vector may further comprise a protein tag which can be optionally removed using an endopeptidase.

[0038] The term "tag" of the present invention refers to a molecule that exhibits an activity or characteristic that can be quantified, and may be a fluorescent molecule including a chemical fluorescent substance (fluoracer) (such as fluorescein) and a polypeptide fluorescent substance (such as fluorescent protein (GFP) or related proteins); or an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, a streptavidin tag, etc. In particular, when an epitope tag is used, a peptide tag consisting of 6 or more amino acid residues may be preferably used, and a peptide tag consisting of 8 to 50 amino acid residues may be more preferably used.

[0039] The term "coronary artery disease (CAD)" of the present invention refers to a disease in which the coronary arteries, which are the main blood vessels of the heart, become narrowed or blocked. Therefore, it may occur due to insufficient oxygen and nutrients in the heart muscle, and this phenomenon is mainly caused by arteriosclerosis. Arteriosclerosis is caused by the accumulation of cholesterol, fat, calcium, etc. on the blood vessel wall, and oxidative stress plays a large role in this process. Oxidative stress can worsen vascular damage and induce inflammation, so coronary artery disease is a disease caused by oxidative stress in myocardial cells that accelerates arteriosclerosis.

[0040] The term "myocardial infarction (MI)" of the present invention may refer to a situation in which the blood supply of the coronary artery is suddenly interrupted, resulting in the arrest of the heart muscle. Oxidative stress can aggravate myocardial cell damage and promote inflammatory response, which can induce cardiac dysfunction.

[0041] The term "heart failure" of the present invention refers to a state in which the heart cannot supply sufficient blood to the body. In this case, oxidative stress in myocardial cells is one of the main causes of heart failure, which worsens cell damage and inflammation and reduces heart function.

[0042] The term "atrial fibrillation (AF)" of the present invention refers to a state in which abnormal electrical signals are generated from the atria of the heart, resulting in a fast and irregular heart rate, which causes the heart to be unable to effectively supply blood to the body. Oxidative stress in myocardial cells is an imbalance in the cell, and oxygen-reactive compounds (free radicals) increase. Such oxidative compounds can damage cells and biomolecules, exacerbate the electrical imbalance of the heart, and increase the possibility of atrial fibrillation.

[0043] Oxidative stress in cardiomyocytes may worsen cardiac damage and inflammation, which may impair the electrical stability of the heart and induce heart disease. Therefore, heart disease can be treated by reducing oxidative stress in cardiomyocytes.

[0044] The term "photodermatitis" of the present invention may refer to a state in which ultraviolet rays or specific substances combined with sunlight cause an inflammatory reaction in the skin. One of the main causes of skin damage caused by exposure to ultraviolet rays is oxidative stress. Oxidative stress can affect dermal fibroblasts, thereby possibly promoting the decomposition of collagen and inflammatory reactions. This process changes the characteristics of the skin and worsens the symptoms of photodermatitis.

[0045] The term "atopic dermatitis" of the present invention is a chronic dermatitis, accompanied by symptoms of dry and itchy skin, characterized by repeated inflammation, which is known to be related to genetic factors, environmental factors, immune system abnormalities, etc., and the stress of dermal fibroblasts may be related to the onset and development of atopic dermatitis. Oxidative stress generates free radicals that damage cells, which can cause inflammatory reactions and low cell function. Dermal fibroblasts are located in the dermis of the skin and play an important role in maintaining the structure and function of the skin. Therefore, when oxidative stress causes the function of dermal fibroblasts to decline, the protective function of the skin may be weakened. As a result, the skin becomes more vulnerable to external stimuli, exacerbating the symptoms of atopic dermatitis. The resulting oxidative stress can promote inflammatory reactions. This inflammatory reaction, as one of the main symptoms of atopic dermatitis, constitutes the reason why dermal fibroblasts cannot function normally. Moreover, cell damage caused by oxidative stress may aggravate the damage to the skin barrier. Damage to the skin barrier makes it easier for substances that cause allergic reactions to penetrate the skin, which further exacerbates the symptoms of atopic dermatitis.

[0046] In the present invention, the expression vector may comprise a nucleotide sequence encoding a peptide consisting of the CPNE7 protein or the amino acids of SEQ ID NO: 1. The vector used in this case may preferably be a plasmid DNA, a phage DNA, etc., more preferably a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an Escherichia coli-derived plasmid (pYG601 BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, adenovirus, vaccinia virus, etc.), virus, etc.), insect virus vectors (baculovirus, etc.), but are not particularly limited thereto as long as the peptide can be produced. The expression vector presents different protein expression amounts and modifications depending on the host cell, so it is preferred to select and use the host cell that best suits the purpose.

[0047] The expression vector provided in the present invention can be introduced into the host for transformation, so that the transformant provided in the present invention can be prepared, and the polynucleotide contained in the expression vector is expressed to be used for the production of the peptide. The transformation can be carried out by a variety of methods, such as the CaCl2 precipitation method, the Hanahan method in which a reducing substance called dimethyl sulfoxide (DMSO (dimethyl sulfoxide)) is used to improve the efficiency in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation, protoplast fusion, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation method, transformation method using polyethylene glycol (PEG), dextran sulfate, liposome and drying / inhibition-mediated transformation method, etc., but as long as the peptide can be produced, it is not particularly limited thereto. Furthermore, the host used in preparing the transformant is not particularly limited as long as it can produce the peptide, but may be bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc.; fungal cells such as Pichia pastoris, etc.; insect cells such as Drosophila, Spodoptera Sf9 cells, etc.; animal cells such as CHO cells, COS cells, NSO cells, 293 cells, human melanoma cells, etc.; or plant cells.

[0048] The transformant can also be used in the method for producing the peptide consisting of the amino acids of sequence number 1 of the present invention as follows. Specifically, the method for producing the peptide consisting of the amino acids of sequence number 1 of the present invention may include: step (a), obtaining a culture by culturing the transformant; and step (b), recovering the peptide of the present invention from the culture.

[0049] The term "culture" of the present invention means a method of growing and developing microorganisms under appropriately artificially adjusted environmental conditions. In the present invention, the method of culturing the transformant can be carried out using a method widely known in the technical field to which the present invention belongs. Specifically, with respect to the culture, continuous culture can be carried out in a batch process or a fed-batch or repeated fed-batch process, but as long as the peptide consisting of the amino acids of sequence number 1 of the present invention can be produced by expressing the peptide, it is not particularly limited thereto.

[0050] The culture medium used for the culture needs to be a conventional culture medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc., under aerobic conditions, and the temperature, pH, etc., and the conditions of the specific strain need to be adjusted in an appropriate manner. As a usable carbon source, a mixed sugar of glucose and xylose is used as the main carbon source, in addition to: sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose; and oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc.; and fatty acids such as palmitic acid, stearic acid, linoleic acid; and alcohols such as glycerol, ethanol; and organic acids such as acetic acid. These substances can be used individually or as a mixture. As usable nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used; 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, etc. can be used. These nitrogen sources can be used alone or in combination. In the culture medium, as a phosphorus source, monopotassium phosphate, dipotassium phosphate and corresponding sodium salts can be included. As usable phosphorus sources, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium salts are included. And as inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate and calcium carbonate can be used. Finally, growth essential substances such as amino acids and vitamins can be added to the substance for use.

[0051] In addition, appropriate precursors may be used in the culture medium. The raw materials may be added to the culture in a batch, fed-batch or continuous manner during the culture process, but are not particularly limited thereto. The pH of the culture may be adjusted by using an alkaline compound such as sodium hydroxide, potassium hydroxide, ammonia, or the like; an acidic compound such as phosphoric acid or sulfuric acid, or the like, in an appropriate manner.

[0052] Furthermore, the generation of bubbles can be suppressed by using a defoaming agent such as fatty acid polyethylene glycol ester. In order 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., and the culture is continued until the production amount of the peptide reaches the maximum value. For this purpose, it can usually be achieved within 10 hours to 100 hours.

[0053] Furthermore, the step of recovering the peptide from the culture may be performed by a method known in the art to which the present invention belongs. Specifically, the recovery method is not particularly limited as long as it is applicable to the recovery of the produced peptide, and preferably, 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) and the like can be used.

[0054] The pharmaceutical composition of the present invention can be prepared into the form of a pharmaceutical composition for treating heart diseases caused by oxidative stress of myocardial cells or skin diseases caused by oxidative stress of dermal fibroblasts, and it also contains appropriate carriers (natural or non-natural carriers), excipients or diluents commonly used in the preparation of pharmaceutical compositions on the basis of the peptide. Specifically, the pharmaceutical composition can be formulated into a sterile injection solution that can be administered to the site of inducing heart disease or skin disease according to the usual method. In the present invention, the carriers, excipients and diluents that can be included in the pharmaceutical composition can be exemplified by lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, etc. When preparing the formulation, commonly used diluents or excipients such as fillers, brighteners, binders, wetting agents, disintegrants, surfactants, etc. can be used for preparation. In particular, it can include sterile aqueous solutions, non-aqueous solvents, suspending agents, emulsions, freeze-dried agents, suppositories, ointments (for example, endodontic transplant materials, etc.), etc. As non-aqueous solvents and suspending agents, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, etc. can be used; injectable esters such as ethyl oleate, etc. can be used. As the base of the suppository, synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, lauric acid glyceride fat, glycerin gelatin, etc. can be used.

[0055] The content of the peptide contained in the pharmaceutical composition of the present invention is not particularly limited, but based on the total weight of the final composition, the peptide may be contained in an amount of 0.0001% to 50% by weight, more preferably 0.01 to 20% by weight.

[0056] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means a sufficient amount for the treatment or prevention of a disease at a reasonable benefit / risk ratio that can be applied 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, gender of the patient, the sensitivity of the patient to the drug, the administration time, administration route and excretion rate of the composition of the present invention used during the treatment, the elements of the drugs used in combination with or containing the composition of the present invention, and other elements known in the medical field. The pharmaceutical composition of the present invention can be administered independently or in combination with a pharmaceutical composition known to be used to treat heart diseases caused by oxidative stress of myocardial cells or skin diseases caused by oxidative stress of dermal fibroblasts. It is important to take all the above factors into consideration and administer the drug in an amount that has no side effects and can achieve the maximum effect with the minimum amount.

[0057] A person skilled in the art of the present invention may determine the dosage of the pharmaceutical composition of the present invention by considering the purpose of use, the degree of disease poisoning, the age, weight, sex, previous illness of the patient, or the type of substance used as an active ingredient. For example, each adult may be administered about 0.1 ng to about 100 mg / kg of the pharmaceutical composition of the present invention, preferably, it can be administered at 1 ng to about 10 mg / kg, and the administration frequency of the composition of the present invention is not particularly limited thereto, but may be administered once a day or divided into doses for multiple administrations. The dosage does not limit the scope of the present invention in any aspect.

[0058] As another embodiment, the present invention provides a method for treating heart disease caused by oxidative stress in cardiomyocytes or skin disease caused by oxidative stress in dermal fibroblasts, which comprises the step of administering the pharmaceutical composition in a pharmaceutically effective amount to an individual other than humans who suffers from heart disease caused by oxidative stress in cardiomyocytes or skin disease caused by oxidative stress in dermal fibroblasts.

[0059] The term "individual" in the present invention refers to mammals that need to treat heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, etc., without limitation, and the mammals include mice, livestock, etc., but exclude humans among individuals suffering from the diseases.

[0060] The pharmaceutical composition for treating a heart disease caused by oxidative stress in cardiomyocytes or a skin disease caused by oxidative stress in dermal fibroblasts of the present invention can be administered by any common route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but can be administered by oral administration, skin administration, intramuscular injection, intravenous injection, subcutaneous injection, respiratory tract administration, rectal administration, and administration to the affected part, etc., depending on the purpose.

[0061] "Oral administration (oral administration)" is a method of taking drugs orally, and drugs in tablet, capsule, or liquid form can usually be used. "Dermal administration" is a method of applying or attaching drugs to the skin, and can be used in the form of cream, gel, patch, etc. "Intramuscular injection" is a method of injecting drugs directly into muscle tissue, and syringes and needles can be mainly used. "Intravenous injection" is a method of injecting drugs directly into blood vessels, and syringes and needles or intravenous catheters can be used. "Subcutaneous injection" is a method of injecting drugs into subcutaneous fat tissue, and can be used for the administration of drugs such as insulin. "Respiratory administration" is a method of allowing drugs to reach the respiratory tract through the mouth or nose, and can be used in the form of inhalers, gas, steam, etc. "Rectal administration" is a method of injecting drugs into the rectum, and can be used in the form of rectal medicine, rectal drops, etc. "Affected area administration" is a method of applying drugs directly to the area that needs treatment, and can be used in the form of cream, gel, or patch depending on the characteristics of the disease.

[0062] As another embodiment, the present invention provides a quasi-pharmaceutical composition for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, comprising the peptide.

[0063] The term "improvement" according to the present invention refers to a parameter related to the condition being treated, for example, all actions that at least reduce the degree of symptoms.

[0064] In the present invention, the improvement can be explained as follows: a pharmaceutical composition comprising the peptide of the present invention as an active ingredient is administered to an individual who needs to treat heart disease caused by oxidative stress in cardiomyocytes or skin disease caused by oxidative stress in dermal fibroblasts to promote the removal of reactive oxygen species in cardiomyocytes or dermal fibroblasts, thereby improving the symptoms of heart disease caused by oxidative stress in cardiomyocytes or skin disease caused by oxidative stress in dermal fibroblasts or obtaining all actions that are beneficially changed.

[0065] The term "quasi-drug" in the present invention refers to items used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases in humans or animals, and whose effects are milder than those of drugs. For example, according to the Pharmaceutical Affairs Law, quasi-drugs refer to items other than those used as drugs, including fiber / rubber products used to treat or prevent human / animal diseases, items that are not instruments or machines and similar items that have slight or no direct effects on the human body, sterilizers / insecticides used to prevent infectious diseases, etc.

[0066] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, and as an example, it may be an ointment, a patch, a powder, a gel, a spray, a tablet or a spray.

[0067] As another embodiment, the present invention provides a health functional food composition for preventing or improving heart diseases caused by oxidative stress of cardiomyocytes or skin diseases caused by oxidative stress of dermal fibroblasts, which comprises the peptide.

[0068] The term "food" of the present invention includes meat, sausage, bread, chocolate, candy, fast food, biscuits, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, rehydration, alcoholic beverages, vitamin complexes, health functional foods and health foods, etc., including all foods in the conventional sense.

[0069] The functional food is the same term as food for special health use (FoSHU), which refers to a food with high medical and medical effects that is processed to effectively present the regulating function of the organism in addition to providing nutrition. Among them, the so-called "function" means to regulate nutrients (to the structure and function of the human body) or to obtain favorable effects in health care purposes (such as physiological effects, etc.). The food of the present invention can be prepared by the method commonly used in the technical field to which the present invention belongs, and the raw materials and ingredients that are usually added in the technical field can be added to prepare it during the preparation. In addition, as for the dosage form of the food, as long as it is a dosage form that can be identified as food, it can be prepared without restriction. The food composition of the present invention has the following advantages: it can be prepared into a variety of dosage forms, and unlike ordinary medicines, food can be used as a raw material, so there are no side effects that may occur when taking medicines for a long time, and it is excellent in portability, so the food of the present invention can be taken as an auxiliary agent for enhancing the effect of preventing or improving heart diseases caused by oxidative stress of myocardial cells or skin diseases caused by oxidative stress of dermal fibroblasts.

[0070] The health food refers to a food that has a positive effect of maintaining or improving health compared to ordinary food, and the health supplement food refers to a food for health supplement purposes. Depending on the situation, the terms health functional food, health food, and health supplement food can be used interchangeably.

[0071] Specifically, the health functional food refers to a food prepared by adding the peptide of the present invention to food materials such as beverages, teas, spices, chewing gums, biscuits, etc., or preparing it as a capsule, powder, suspension, etc. When the food is ingested, it brings specific effects on health. However, unlike ordinary medicines, since food is used as a raw material, there is no advantage of side effects that may occur when taking medicines for a long time.

[0072] The food composition of the present invention can be ingested daily, and therefore can be expected to have a good effect in preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, and can therefore be used very effectively.

[0073] The food composition may further include a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the technical field to which the present invention belongs may be used.

[0074] Furthermore, the food composition may include additives that can be commonly used in food compositions to improve smell, taste, vision, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folic acid, pantothenic acid, etc. may be included. Furthermore, minerals such as zinc (Zn), iron (Fe), calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. may be included. Furthermore, amino acids such as lysine, tryptophan, cysteine, valine, etc. may be included.

[0075] Moreover, the food composition may contain food additives, such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high-efficiency bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar pigments, etc.), colorants (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (sweetness, cyclamates, saccharin, sodium, etc.), spices (vanillin, lactones, etc.), expanders (alum, D-potassium hydrogen tartrate, etc.), enhancers, emulsifiers, thickeners (paste), film-forming agents, glue bases, foam inhibitors, solvents, improvers, etc. The additives may be selected and used in appropriate amounts according to the type of food.

[0076] Peptide of the present invention can be directly added or used together with other foods or food ingredients, and can be suitably used according to conventional methods.Can suitably determine the mixed amount of active ingredient according to its purpose of use (prevention, health or therapeutic treatment).Usually, when preparing food or beverage, relative to food or beverage, food composition of the present invention can add below 50 weight portions, specifically, can add the amount below 20 weight portions.But, in the case of long-term intake for the purpose of health and hygiene, can comprise the amount below the described scope, owing to there is no problem aspect safety, can also use the amount above the described scope as active ingredient.

[0077] As an example of the food composition of the present invention, it can be used as a health drink composition. In the case, it can contain a variety of flavoring agents or natural carbohydrates as added ingredients, just like general beverages. The natural carbohydrates can 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, erythritol, etc. Sweeteners can use natural sweeteners, such as thaumatin and stevia extract; synthetic sweeteners, such as saccharin and aspartame, etc. The ratio of the natural carbohydrates in each 100 mL of the health drink composition of the present invention can generally be about 0.01g to 0.04g, specifically about 0.02g to 0.03g.

[0078] In addition to the above, the health drink composition can include multiple nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid, pectinate, alginic acid, alginate, organic acid, protective colloid thickener, pH adjusting agent, stabilizer, preservative, glycerine, ethanol or carbonating agent etc. In addition, the pulp used to prepare natural fruit juice, fruit juice beverage or vegetable beverage can be included. This composition can be used independently or in combination. Although the ratio of this additive is not particularly important, the health drink composition of the present invention of every 100 weight portions is selected usually in the range of 0.01 to 0.1 weight portion.

[0079] As for the food composition of the present invention, if it can show the effect of preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts, the peptide of the present invention can be included in various weight %, specifically, based on the total weight of the food composition, it can include 0.00001 to 100 weight % or 0.01 to 80 weight % of the peptide of the present invention, but is not limited to this.

[0080] Effects of the Invention

[0081] The present invention can provide a composition for removing active oxygen in cells containing a CPNE7 protein or a peptide derived from a CPNE7 protein.

[0082] Another object of the present invention is to provide a polynucleotide encoding the peptide.

[0083] Another object of the present invention is to provide an expression vector comprising the polynucleotide.

[0084] Another object of the present invention is to provide a pharmaceutical composition comprising the peptide for preventing or treating heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts.

[0085] Another object of the present invention is to provide a quasi-pharmaceutical composition comprising the peptide for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts.

[0086] Another object of the present invention is to provide a health functional food composition comprising the peptide for preventing or improving heart diseases caused by oxidative stress in cardiomyocytes or skin diseases caused by oxidative stress in dermal fibroblasts.

[0087] The effects of the present invention are not limited to the above-mentioned contents, and a person having ordinary knowledge in the technical field to which the present invention belongs can clearly understand other effects not mentioned from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 As a graph comparing active oxygen levels using the cell permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH), the results show that when Cpne7 was reduced by ShCpne7 conversion, the active oxygen level increased by about 2 times or more compared to the control group.

[0089] Figure 2 The results are a comparison of active oxygen levels using the cell permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH).

[0090] Figure 3 The results are obtained by measuring the amount of living cells using WST-8 (High Sensitive Water Soluble Tetrazolium Salt).

[0091] Figure 4 The results are the evaluation results of the effect of increased reactive oxygen species on odontoblast production in Cpne7 knockout mice (DNA damage) and the comparative confirmation results of the degree of DNA damage by immunofluorescence staining of γ-H2A.X, a DNA damage marker protein.

[0092] Figure 5 As a result of evaluating the effect of increased reactive oxygen species on odontoblast production (cell aging) in Cpne7 knockout mice, the degree of cell aging was compared by staining for β-galactosidase (B-gal), a cell aging marker enzyme.

[0093] Figure 6 This is the result of evaluating the effect of increased reactive oxygen species on odontoblast production (histological analysis) in Cpne7 knockout mice.

[0094] Figure 7 These are the results of confirming the ability of CPNE7 recombinant protein (rCPNE7) to remove active oxygen species.

[0095] Figure 8The results were to confirm the ability of CPNE7 recombinant protein (rCPNE7) to remove reactive oxygen species by comparing reactive oxygen species levels using the cell permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH).

[0096] Fig. 9 The results are as follows: the degree of DNA damage was compared and confirmed by immunofluorescence staining of γ-H2A.X, a DNA damage marker protein, and the ability of CPNE7 recombinant protein (rCPNE7) to remove active oxygen species was confirmed by using this method.

[0097] Fig.10 The results are as follows: the degree of cell aging was compared by staining for Beta-Galactosidase (B-gal), a cell aging marker enzyme, and the ability of the CPNE7 recombinant protein (rCPNE7) to remove active oxygen was confirmed by using this.

[0098] Fig.11 As a result of measuring the amount of living cells using WST-8 (High Sensitive Water Soluble Tetrazolium Salt), the cell viability of hDPCs at different concentrations of active oxygen was confirmed.

[0099] Fig.12 These are the results of confirming the ability to remove active oxygen species at different concentrations of a functional peptide (Selcopintide) derived from the CPNE7 protein.

[0100] Fig.13 The results were obtained by comparing the levels of reactive oxygen species using the cell permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH), and confirming the reactive oxygen species removal ability of a functional peptide (Selcopintide) derived from the CPNE7 protein at different concentrations.

[0101] Fig.14The results are to compare the level of reactive oxygen species in hDPCs cells using the mRNA level of endogenous antioxidant enzymes of Selcopintide by RT-PCR, and to confirm the reactive oxygen species removal ability of the functional peptide (Selcopintide) derived from the CPNE7 protein at different concentrations.

[0102] Fig.15 The results are as follows: the degree of DNA damage was compared and confirmed by immunofluorescence staining of γ-H2A.X, a marker protein for DNA damage, and the ability of Selcopintide to remove active oxygen species was confirmed by using this method.

[0103] Fig.16 The results are obtained by measuring the amount of living cells using WST-8 (High Sensitive Water Soluble Tetrazolium Salt).

[0104] Fig.17 After treating with H2O2, which induces intracellular reactive oxygen species, and treating with Selcopintide, the cells were confirmed under an optical microscope and the amount of living cells was measured using WST-8 (High Sensitive Water Soluble Tetrazolium Salt). The results confirmed the ability of Selcopintide to remove reactive oxygen species in cardiomyocytes.

[0105] Fig.18 The results are as follows: the degree of cell aging was compared by staining for Beta-Galactosidase (B-gal), a cell aging marker enzyme, and the ability of Selcopintided to remove active oxygen in cardiomyocytes was confirmed by using this.

[0106] Fig.19 The results are as follows: the amount of living cells was measured using WST-8 (High Sensitive Water Soluble Tetrazolium Salt), and the active oxygen-removing ability of Selcopintide in dermal fibroblasts was confirmed by using the measurement.

[0107] Fig. 20 The results are to confirm the active oxygen scavenging ability of Selcopintide in dermal fibroblasts.

[0108] Fig.21The results are as follows: the degree of cell aging was compared by staining for Beta-Galactosidase (B-gal), a cell aging marker enzyme, and the ability of Selcopintide to remove active oxygen in dermal fibroblasts was confirmed by using this. DETAILED DESCRIPTION

[0109] If you refer to the following and Figure 1 The purpose and effect of the present invention and the technical structure used to achieve the same will be clear from the detailed description of the embodiments. In describing the present invention, when it is judged that the detailed description of the known function or structure may unnecessarily confuse the gist of the present invention, the detailed description will be omitted. Moreover, the terms described below, as terms defined in consideration of the functions in the present invention, may vary according to the intention or convention of the user or operator.

[0110] However, the present invention is not limited to the embodiments disclosed below, and can be embodied in various forms that are different from each other. However, this embodiment is provided to make the disclosure of the present invention more complete and to fully explain the scope of the invention to people with ordinary knowledge in the technical field to which the present invention belongs, and the present invention is only defined by the scope of the claims. Therefore, this definition should be made based on the entire content of this specification.

[0111] Hereinafter, embodiments of the present invention will be described in more detail.

[0112] Example 1. Comparison of reactive oxygen species levels using the cell permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH)

[0113] Human dental pulp cells (hDPCs) were cultured in an α-MEM medium containing 10% FBS at 5% CO 2 and 37° C.

[0114] Then, 4x10 5 The hDPCs cells were inoculated with the cultured cells at a number of cells / dish, and after culturing for 24 hours, the cells were transfected with Lipofectamine Plus TM The reagents are introduced into the cultured cells to transform the Shcon vector and the ShCpne7 vector.

[0115] The transformed hDPCs cells were cultured at 3×10 3After the cells were re-seeded in a 96-well plate and cultured for 24 hours, the DCFDA substances were treated respectively, and the reactive oxygen levels were measured at Ex / Em=485 / 535nm using an ELISA reader 40 minutes later. At this time, a control group without any treatment and a Shcon vector were used as the control group.

[0116] refer to Figure 1 When Cpne7 was reduced by ShCpne7 conversion, the level of reactive oxygen species increased by more than 2 times compared with the control group. This indicates that Cpne7 is effective in maintaining the level of reactive oxygen species at a normal level in hDPCs cells.

[0117] Example 2. Comparison of reactive oxygen species levels using the cell permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH)

[0118] The DCFDA substances were treated on the mandibular molar tissue sections of 6-month-old mice in which the Cpne7 gene was knocked out, and the reactive oxygen levels were measured by confocal microscopy 40 minutes later. At this time, mandibular molars of 6-month-old BL6 mice were used as a control group.

[0119] When the Cpne7 gene was knocked out, it was confirmed that the level of reactive oxygen species in odontoblasts in vivo increased compared with the control group, similar to the in vitro results ( Figure 2 ).

[0120] These results confirmed that Cpne7 is a factor that maintains the level of active oxygen at a normal level in the body including odontoblasts.

[0121] Example 3. Verification of oxidative stress susceptibility by knockdown of Cpne7 in odontoblasts

[0122] In a 96-well plate, 3x10 3 hDPCs cells were inoculated with the number of cells / well and cultured for 24 hours, and then treated with hydrogen peroxide (H2O2) at 50uM and 100uM for 24 hours. After the WST-8 solution was added to each well and reacted for 1 hour, the absorbance was measured and compared at 450nm using an ELISA reader.

[0123] According to the treatment results, in the group with knockdown of Cpne7 gene (ShCpne7), when H2O2 concentration was 100uM, the cell survival rate was significantly reduced compared with the control group ( Figure 3 ). Therefore, it can be confirmed that Cpne7 is involved in maintaining the level of reactive oxygen species at a normal level in hDPCs cells.

[0124] Example 4. Comparative confirmation of the degree of DNA damage by immunofluorescence staining of γ-H2A.X, a DNA damage marker protein

[0125] From the said Figure 1 and Figure 2 The results confirmed that Cpne7 is involved in maintaining normal levels of reactive oxygen species, and in particular, when Cpne7 was knocked out, reactive oxygen species increased. Based on this, it was confirmed whether the increase in reactive oxygen species increases DNA damage in odontoblasts.

[0126] Immunofluorescence staining using γ-H2A.X antibody was performed on tissue sections of mandibular molars of 6-month-old Cpne7 knockout mice to compare the intensity. At this time, tissue sections of mandibular molars of 6-month-old BL6 mice were used as a control group.

[0127] As in Figure 4 In the study, it was confirmed that DNA damage accumulated in odontoblasts of Cpne7 knockout mice in which reactive oxygen species were increased.

[0128] Based on this analysis, the increase in reactive oxygen species caused by Cpne7 knockout will induce DNA damage.

[0129] Example 5. Comparison of the degree of cell aging by staining for Beta-Galactosidase (B-gal), a cell aging marker enzyme

[0130] From the said Figure 1 , Figure 2 and Figure 4 The results confirmed that the increased reactive oxygen species associated with Cpne7 knockout induced DNA damage, and based on this, we examined whether the induced DNA damage could promote cell aging.

[0131] In a 35 mm culture dish, 1x10 5After the transformed hDPCs were inoculated with a cell number and cultured for 24 hours, the α-MEM medium containing 5% FBS, 50ug / ml ascorbic acid and 10mM β-glycerophosphate was replaced every two days to allow cell differentiation until day 21. Differentiated odontoblasts on days 0, 4, 7, 14 and 21 were fixed with 4% paraformaldehyde and B-gal staining was performed in a CO2-free 37°C incubator for 24 hours.

[0132] Furthermore, B-gal staining was performed for 24 hours on mandibular molar tissue sections of 6-month-old Cpne7 knockout mice in which reactive oxygen species and DNA damage were confirmed. Figure 5 As shown, it was confirmed that in odontoblasts in which the Cpne7 gene was knocked down or knocked out, more β-gal-positive (B-gal positive) cells were found than in the control group.

[0133] Therefore, it was confirmed that the increase in reactive oxygen species caused by Cpne7 knockout induces DNA damage and promotes cell aging.

[0134] Example 6. Comparison of histomorphology of mandibular molars of 6-month-old and 12-month-old Cpne7 knockout mice

[0135] Hematoxylin and Eosin (H&E) staining was performed on tissue sections of mandibular molars of 6-month-old and 12-month-old Cpne7 knockout mice.

[0136] From the said Figure 1 , Figure 2 , Figure 3 and Figure 4 It was confirmed that in Cpne7 knockout odontoblasts, the induction of DNA damage and cell senescence were promoted along with the increase of reactive oxygen species.

[0137] Based on the results of histomorphological comparison of the mandibular molars of 6-month-old and 12-month-old Cpne7 knockout mice, it was confirmed that the odontoblasts in the mandibular molars of 6-month-old Cpne7 knockout mice not only showed irregular arrangement and abnormal morphology of the cell nuclei, but also pathological bone-like hard tissue was formed in the pulp tissue.

[0138] In the tooth tissue of 12-month-old Cpne7 knockout mice, it was confirmed that more pathological bone-like hard tissue was formed in the pulp tissue, most of the odontoblasts died, and the state of the remaining odontoblasts was not healthy compared with the control group.

[0139] Therefore, it was confirmed that the increase in reactive oxygen species caused by Cpne7 knockout induces DNA damage and cell senescence in odontoblasts, leading to a pathological state.

[0140] Example 7. After treating H2O2, which induces intracellular reactive oxygen species, and treating CPNE7 recombinant protein, the cells were confirmed under an optical microscope.

[0141] In a 35 mm culture dish, 1x10 5 hDPCs were inoculated in the manner of 100 cells per day, and after culturing for 24 hours, H2O2 was treated at a concentration of 5uM, 100uM, 1mM or 2mM, and then 100ng / ml of recombinant CPNE7 protein was treated, and then cultured for 24 hours. After the culture was completed, the cultured cells were washed with PBS and observed under an optical microscope. At this time, hDPCs cultured without the treatment of the recombinant CPNE7 protein (rCPNE7) were used as a control group.

[0142] like Figure 7 As shown, no change was observed in H2O2 at concentrations below 100uM, and cell death due to oxidative stress was observed at concentrations above 1mM. In contrast, a higher cell survival rate was observed in the treated group compared to the control group for the recombinant CPNE7 protein. Based on this analysis, the CPNE7 protein has the effect of removing active oxygen species.

[0143] Example 8. Comparison of reactive oxygen species levels using the cell permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH)

[0144] 1x10 5 hDPCs were inoculated in culture according to the cell number and cultured for 24 hours. Then, they were treated with 1 mM H2O2 and 100 ng / ml rCPNE7 and photographed with a confocal microscope for about 1 hour.

[0145] refer to Figure 8It was confirmed that the amount of active oxygen reached a maximum value about 30 minutes after treatment with 1 mM H2O2, and then decreased significantly about 1 hour later. This confirmed the ability of CPNE7 protein to remove active oxygen.

[0146] Example 9. Comparison of DNA damage levels by immunofluorescence staining of γ-H2A.X, a DNA damage marker protein

[0147] From the said Figure 8 It was confirmed that CPNE7 recombinant protein removed excessive reactive oxygen species in hDPCs, and the extent of DNA damage was determined based on this.

[0148] 1x10 5 hDPCs were inoculated in culture according to the cell number and cultured for 24 hours. Then, they were treated with 1 mM H2O2 and 100 ng / ml rCPNE7. After 24 hours, the cells were fixed with 4% paraformaldehyde.

[0149] Immunofluorescence staining using γ-H2A.X antibody was performed in fixed cells, and the intensities were compared.

[0150] refer to Fig. 9 According to the comparison results, in the group treated with 1mM H2O2 to give oxidative stress, a considerable amount of DNA damage was confirmed to be induced, while in the group treated with 100ng / ml CPNE7 recombinant protein, the accumulation of DNA damage was confirmed to be significantly reduced. This confirms that CPNE7 protein removes excessive active oxygen, thereby preventing or repairing DNA damage.

[0151] Example 10. Comparison of the degree of cell aging by staining for Beta-Galactosidase (B-gal), a cell aging marker enzyme

[0152] From the said Figure 8 and Fig. 9 , confirmed that the removal of excessive reactive oxygen species by CPNE7 recombinant protein in hDPCs can prevent or repair DNA damage, and wanted to confirm the impact of this on cell aging.

[0153] To do this, place 1x10 5 hDPCs were inoculated in culture according to the cell number and treated with 1 mM H2O2 and 100 ng / ml rCPNE7 after 24 hours of culture. The cells were fixed with 4% paraformaldehyde after 12 hours or 24 hours.

[0154] Then, B-gal staining was performed in fixed cells for 24 hours, and the degree of cell senescence was compared.

[0155] refer to Fig.10 , after 12 hours of treatment with 1mM H2O2, B-gal-positive hDPCs cells were confirmed, and more were confirmed after 24 hours. In contrast, compared with the H2O2 alone treatment group, a significant decrease in B-gal-positive hDPCs cells was confirmed in the CPNE7 recombinant protein treatment group. This confirms that the active oxygen removal ability of CPNE7 protein can prevent or repair DNA damage, thereby inhibiting cell aging.

[0156] Example 11. Measurement of the amount of living cells using WST-8 (High Sensitive Water Soluble Tetrazolium Salt)

[0157] In a 96-well plate at 3x10 3 hDPCs cells were inoculated with 0uM (control group), 50uM, 100uM, 200uM, 500uM, 1mM and 2mM hydrogen peroxide (H2O2) for 24 hours after culturing for 24 hours. After the WST-8 solution was added to each well and reacted for 1 hour, the absorbance was measured and compared at 450nm using a microplate reader.

[0158] like Fig.11 As shown, it was confirmed that the cell survival rate decreased with the increase of H2O2 concentration, and similar cell survival rates were confirmed when the H2O2 concentration was 500uM or higher. Then, the H2O2 concentration of 1mM was selected as the oxidative stress-inducing concentration.

[0159] Example 12. Synthesis of peptides for removing intracellular reactive oxygen species

[0160] The present inventors synthesized a peptide (SEQ ID NO. 1) showing an effect of removing intracellular active oxygen species by the 9-fluorenylmethyloxycarbonyl (Fmoc) method, and synthesized peptides of each group (Tables 1 to 12) by substituting amino acids of the synthesized peptide.

[0161] N-KYQRRKKNKY-C (SEQ ID NO. 1)

[0162] First, the peptide of Group 1 is the peptide of Sequence No. 1, or is synthesized by substituting lysine or arginine for amino acids 5 to 7 of the peptide of Sequence No. 1 (Table 1).

[0163]

Table 1

[0164] Group 1 peptides

[0165]

[0166] Then, the amino acids at positions 5 to 7 of the peptide of SEQ ID NO: 1 were substituted with lysine or arginine, and the amino acid at position 8 was substituted with serine, thereby synthesizing peptides of Group 2 (Table 2).

[0167]

Table 2

[0168] Group 2 peptides

[0169]

[0170] Then, the amino acids at positions 5 to 7 of the peptide of SEQ ID NO: 1 were substituted with lysine or arginine, and the amino acid at position 9 was substituted with tyrosine, thereby synthesizing peptides of Group 3 (Table 3).

[0171]

Table 3

[0172] Group 3 peptides

[0173]

[0174] Then, the amino acids at positions 5 to 7 of the peptide of sequence number 1 were substituted with lysine or arginine, the amino acid at position 8 was substituted with serine, the amino acid at position 9 was substituted with tyrosine, and the amino acid at position 10 was substituted with lysine, thereby synthesizing peptides of group 4 (Table 4).

[0175]

Table 4

[0176] Group 4 peptides

[0177]

[0178] Then, the amino acid at position 3 of the peptide of sequence number 1 was substituted with arginine, the amino acid at position 4 was substituted with glutamine, and the amino acids at positions 5 to 7 were substituted with lysine or arginine, thereby synthesizing peptides of group 5 (Table 5).

[0179]

Table 5

[0180] Group 5 peptides

[0181]

[0182] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced with arginine, the amino acid at position 4 was replaced with glutamine, the amino acids at positions 5 to 7 were replaced with lysine or arginine, and the amino acid at position 8 was replaced with serine, thereby synthesizing peptides of group 6 (Table 6).

[0183]

Table 6

[0184] Group 6 peptides

[0185]

[0186] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by arginine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 7 (Table 7).

[0187]

Table 7

[0188] Group 7 peptides

[0189]

[0190] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by arginine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 8 was replaced by serine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 8 (Table 8).

[0191]

Table 8

[0192] Group 8 peptides

[0193]

[0194]

[0195] Then, the amino acid at position 3 of the peptide of sequence number 1 was substituted with lysine, the amino acid at position 4 was substituted with glutamine, and the amino acids at positions 5 to 7 were substituted with lysine or arginine, thereby synthesizing peptides of group 9 (Table 9).

[0196]

Table 9

[0197] Group 9 peptides

[0198]

[0199] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, and the amino acid at position 8 was replaced by serine, thereby synthesizing peptides of group 10 (Table 10).

[0200]

Table 10

[0201] Group 10 peptides

[0202]

[0203]

[0204] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 11 (Table 11).

[0205]

Table 11

[0206] Group 11 peptides

[0207]

[0208] Finally, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 8 was replaced by serine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 12 (Table 12).

[0209]

Table 12

[0210] Group 12 peptides

[0211]

[0212] Example 13. Confirmation of the ability to remove active oxygen species at different concentrations of a functional peptide (Selcopintide) derived from CPNE7 protein

[0213] Fig.12 The results are as follows: cells were confirmed on an optical microscope and the amount of live cells was measured using WST-8 (High Sensitive Water Soluble Tetrazolium Salt) after treatment with H2O2 that induces intracellular reactive oxygen species and Selcopintide (SEQ ID NO: 96).

[0214] In a 6-well plate, 1x10 5 hDPCs were inoculated with 1 mM hydrogen peroxide (H2O2) after 24 hours of culture, 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, hDPCs without any treatment were used.

[0215] Compared with the control group, a considerable amount of odontoblast cell death was confirmed in the 1mM H2O2 treatment group. On the contrary, compared with the H2O2 single treatment group, the cell survival rate in the Selcopintide treatment group was higher, especially in the 1ug / ml and 10ug / ml treatment groups ( Fig.12 ).

[0216] In a 96-well plate, 3x10 3 hDPCs were inoculated with 1 mM hydrogen peroxide (H2O2) after 24 hours of culture, 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, hDPCs without any treatment were used.

[0217] After the WST-8 solution was added to each well and reacted for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader. Compared with the control group, the cell survival rate was significantly reduced in the H2O2 treatment group, but compared with the H2O2 single treatment group, the cell survival rate was significantly higher in the 1ug / ml and 10ug / ml Selcopintide treatment groups ( Fig.12 ).

[0218] Thus, the active oxygen scavenging ability of the functional peptide (Selcopintide) derived from the CPNE7 protein was confirmed.

[0219] Example 14. Comparison of reactive oxygen species levels using the cell permeant reagent 2',7'-dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA, DCFH-DA, and DCFH) and confirmation of the reactive oxygen species removal ability of the functional peptide (Selcopintide) derived from the CPNE7 protein at different concentrations

[0220] 1x10 5 hDPCs were inoculated in culture in the manner of cell number, and after culturing for 24 hours, they were treated with 1 mM H2O2 and 10 ug / ml Selcopintide, and photographed with a confocal microscope for about 1 hour.

[0221] refer to Fig.13It was confirmed that after treatment with 1 mM H2O2, the amount of active oxygen showed a maximum value for about 30 minutes, and then showed a significant decrease for about 1 hour. This confirmed the active oxygen removal potential of Selcopintide.

[0222] Example 15. Confirmation of mRNA levels of endogenous antioxidant enzymes of Selcopintide in hDPCs by RT-PCR

[0223] In a 60 mm dish, 4 x 10 5 hDPCs were inoculated in culture according to the cell number and treated with 1 mM H2O2 after 24 hours of culture. After treatment with CPNE7 recombinant protein (100 ng / ml) and Selcopintide (10 ug / ml), the mRNA levels of Sod1, Sod2, Sod3, catalase, Gpx7, Hmox1 and Txrnd1 were confirmed after 30 minutes and 1 hour.

[0224] refer to Fig.14 It was confirmed that after 30 minutes of treatment with CPNE7 recombinant protein and Selcopintide, the endogenous antioxidant enzymes Sod1, Sod2, and catalase increased significantly compared with the H2O2 alone treatment group. After one hour of treatment with CPNE7 recombinant protein and Selcopintide, it was confirmed that Hmox1 and Txrnd1, which inhibit the formation of endogenous antioxidant enzymes Sod1, Gpx7 and OH free radicals, increased significantly.

[0225] This confirmed that Selcopintide can remove active oxygen by regulating endogenous antioxidant enzymes similar to the CPNE7 protein.

[0226] Example 16. Comparative confirmation of the extent of DNA damage by immunofluorescence staining of γ-H2A.X, a DNA damage marker protein

[0227] As observed above, it was confirmed that the CPNE7 recombinant protein can prevent or repair DNA damage in hDPCs by removing excessive reactive oxygen species, thereby inhibiting cell aging.

[0228] 1x10 5 hDPCs were inoculated in culture according to the cell number, and after culturing for 24 hours, they were treated with 1 mM H2O2 and 10 ug / ml Selcopintide, and fixed with 4% paraformaldehyde after 24 hours.

[0229] Immunofluorescence staining using γ-H2A.X antibody was performed on fixed cells, and the intensities were compared.

[0230] refer to Fig.15 It was confirmed that in the group treated with 1 mM H2O2 to impart oxidative stress, a considerable amount of DNA damage was induced. On the contrary, in the group treated with 10 ug / ml Selcopintide, the accumulation of DNA damage was significantly reduced.

[0231] This confirmed that Selcopintide can prevent or repair DNA damage by removing excess reactive oxygen species similarly to the CPNE7 protein.

[0232] Example 17-1. Measurement of the amount of living cells using WST-8 (High Sensitive Water Soluble Tetrazolium Salt)

[0233] In the above-mentioned study on odontoblasts, it was confirmed that CPNE7 protein and Selcopintide have the ability to remove active oxygen. Similar to odontoblasts, it is known that the peptides also have the ability to remove active oxygen in cardiomyocytes differentiated from MSCs (mesenchymal stem cells).

[0234] In a 96-well plate at 3x10 3 Cell number / well inoculated cultured cardiomyocytes (rat cardiomyocytes (H9C2 cellline)), and after culturing for 24 hours, treated with hydrogen peroxide (H2O2) for 24 hours at 0uM (control group (Control)), 50uM, 100uM, 200uM, 500uM, 1mM and 2mM. After adding the WST-8 solution to each well and reacting for 1 hour, the absorbance was measured and compared at 450nm using an ELISA reader.

[0235] like Fig.16 As shown, it was confirmed that the cell viability decreased with the increase in H2O2 concentration, and similar cell viability was confirmed when the H2O2 concentration was 500uM or higher.

[0236] Thereafter, a concentration of 1 mM H2O2 was selected as the oxidative stress-imparting concentration in H9C2 cells.

[0237] Example 17-2. After treating with H2O2 to induce intracellular reactive oxygen species and treating with Selcopintide, the cells were confirmed on an optical microscope and the amount of living cells was measured using WST-8 (High Sensitive WaterSoluble Tetrazolium Salt)

[0238] In a 6-well plate, 1x10 5 H9C2 cells were inoculated and cultured for 24 hours, and then treated with 1mM hydrogen peroxide (H2O2), and then treated with Selcopintide at concentrations of 1ug / ml, 10ug / ml, 100ug / ml, and 500ug / ml for 24 hours. H9C2 cells without any treatment were used as a control group.

[0239] Compared with the control group, a considerable amount of cardiomyocyte cell death was confirmed in the 1 mM H2O2 treatment group. In contrast, the cell survival rate was higher in the Selcopintide treatment group than in the H2O2 alone treatment group ( Fig.17 ).

[0240] In a 96-well plate at 3x10 3 H9C2 cells were inoculated and cultured for 24 hours, and then treated with 1mM hydrogen peroxide (H2O2), and then treated with Selcopintide at concentrations of 1ug / ml, 10ug / ml, 100ug / ml, and 500ug / ml for 24 hours. H9C2 cells without any treatment were used as a control group.

[0241] After the WST-8 solution was added to each well and reacted for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader.

[0242] Compared with the control group, the cell survival rate was significantly reduced in the H2O2 treatment group, but compared with the H2O2 treatment group alone, the cell survival rate was significantly higher in the Selcopintide treatment groups at 1ug / ml, 10ug / ml, 100ug / ml and 500ug / ml ( Fig.17 ).

[0243] This confirmed that the functional peptide (Selcopintide) derived from the CPNE7 protein also has the ability to remove active oxygen in cardiomyocytes.

[0244] Example 18. Comparison of the degree of cell aging by staining Beta-Galactosidase (B-gal), a cell aging marker enzyme

[0245] In the Fig.17 It was confirmed that Selcopintide is effective in removing excessive reactive oxygen species in H9C2 cells, and the effect of this on cell aging was determined.

[0246] In a 6-well plate, 1x10 5 H9C2 cells were inoculated according to the cell number and treated with 1mM H2O2 and Selcopintide at 1ug / ml, 10ug / ml, 100ug / ml and 500ug / ml after 24 hours. Cells were fixed with 4% paraformaldehyde.

[0247] Then, B-gal staining was performed in fixed cells for 24 hours, and the degree of cell senescence was compared.

[0248] After 24 hours of treatment with 1 mM H2O2, a large number of B-gal-positive H9C2 cells were confirmed. In contrast, it was confirmed that the number of B-gal-positive H9C2 cells in the Selcopintide treatment group was significantly reduced compared to the H2O2 single treatment group.

[0249] This analysis showed that the active oxygen removal ability of Selcopintide can inhibit cardiomyocyte aging.

[0250] Example 19. Measurement of the amount of living cells using WST-8 (High Sensitive Water Soluble Tetrazolium Salt)

[0251] In the above studies on odontoblasts and cardiomyocytes, it was confirmed that Selcopintide has the ability to remove active oxygen. Next, similar to odontoblasts and cardiomyocytes, it was determined whether dermal fibroblasts, which are known to be differentiated from MSCs (mesenchymal stem cells), also have the ability to remove active oxygen.

[0252] In a 96-well plate at 3x10 3 Cell number / well cultured dermal fibroblasts (CCD986SK) were inoculated and cultured for 24 hours, and hydrogen peroxide (H2O2) was treated for 24 hours at 0uM (control group (Control)), 50uM, 100uM, 200uM, 500uM, 1mM and 2mM. After the WST-8 solution was added to each well and reacted for 1 hour, the absorbance was measured and compared at 450nm using an ELISA reader.

[0253] like Fig.19 As shown, it was confirmed that the cell viability decreased as the H2O2 concentration increased, and similar cell viability was confirmed when the H2O2 concentration was 500uM or higher.

[0254] Thereafter, a concentration of 1 mM H2O2 was selected as the oxidative stress-imparting concentration in CCD986SK cells.

[0255] Example 20. Confirmation of cells on an optical microscope and measurement of the amount of live cells using WST-8 (High Sensitive Water Soluble Tetrazolium Salt) after treatment with H2O2 that induces intracellular reactive oxygen species and treatment with Selcopintide

[0256] In a 6-well plate, 1x10 5 CCD986SK cells were inoculated and cultured at the number of cells / well. After 24 hours of culture, 1mM hydrogen peroxide (H2O2) was treated, and then Selcopintide was treated at concentrations of 1ug / ml, 10ug / ml, 100ug / ml, and 500ug / ml for 24 hours. CCD986SK cells without any treatment were used as the control group.

[0257] Compared with the control group, a considerable amount of cell death of dermal fibroblasts was confirmed in the group treated with 1 mM H2O2. In contrast, compared with the group treated with H2O2 alone, the cell survival rate was higher in the group treated with Selcopintide, and was significantly higher in the group treated with 1 ug / ml and 10 ug / ml Selcopintide ( Fig. 20 ).

[0258] In a 96-well plate at 3x10 3 CCD986SK cells were inoculated and cultured for 24 hours, and then treated with 1mM hydrogen peroxide (H2O2), and then treated with Selcopintide at concentrations of 1ug / ml, 10ug / ml, 100ug / ml, and 500ug / ml for 24 hours. CCD986SK cells without any treatment were used as a control group.

[0259] After the WST-8 solution was added to each well and reacted for 1 hour, the absorbance was measured and compared at 450 nm using a microplate reader.

[0260] Although the cell survival rate was significantly reduced in the H2O2-treated group compared to the control group, the cell survival rate was significantly higher in the Selcopintide-treated groups at 1ug / ml, 10ug / ml, 100ug / ml, and 500ug / ml compared to the H2O2-treated group ( Fig. 20 ).

[0261] Therefore, it was confirmed that the functional peptide (Selcopintide) derived from the CPNE7 protein has the ability to remove active oxygen in dermal fibroblasts.

[0262] Example 21. Comparison of the degree of cell aging by staining Beta-Galactosidase (B-gal), a cell aging marker enzyme

[0263] As described Fig. 20 As shown, it was confirmed that Selcopintide has an effect on removing excessive reactive oxygen species in CCD986SK cells, and the effect of this on cell aging was examined.

[0264] In a 6-well plate, 1x10 5 CCD986SK cells were inoculated in culture according to the cell number, and after 24 hours of culture, they were treated with 1mM H2O2 and Selcopintide at 1ug / ml, 10ug / ml, 100ug / ml and 500ug / ml. After 24 hours, the cells were fixed with 4% paraformaldehyde.

[0265] B-gal staining was performed in fixed cells for 24 hours and the degree of cell senescence was compared.

[0266] After 24 hours of treatment with 1 mM H2O2, a large number of B-gal-positive CCD986SK cells were confirmed. In contrast, it was confirmed that the number of B-gal-positive CCD986SK cells in the Selcopintide treatment group was significantly reduced compared to the H2O2 single treatment group.

[0267] Based on this analysis, the ROS removal ability of Selcopintide can inhibit the aging of dermal fibroblasts.

[0268] From the examples observed above, it can be seen that CPNE7 protein and functional peptides derived from CPNE7 protein (Selcopintide) prevent or treat dental pulp diseases, myocardial infarction, skin inflammation, etc. caused by excessive reactive oxygen (H2O2), and thus can be used to prevent individual aging.

[0269] The preferred embodiments of the present invention are disclosed in this specification and the accompanying drawings. Although specific terms are used, they are used in a general sense only to easily explain the technical content of the present invention and to help understand the invention, and are not used to limit the scope of the present invention. In addition to the embodiments disclosed herein, other variations based on the technical ideas of the present invention may also be implemented, which is obvious to a person skilled in the art of the present invention.

Claims

1. A composition for removing reactive oxygen species in cells, wherein the cells contain CPNE7 protein or a peptide consisting of amino acids in any one of SEQ ID NOs. 1 to 96.

2. The composition for removing intracellular active oxygen according to claim 1, characterized in that: The cells are dental pulp cells, cardiomyocytes or dermal fibroblasts.

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

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

5. An expression vector comprising the polynucleotide of claim 4.

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

7. The composition according to claim 6, wherein The pharmaceutical composition comprises a polypeptide formed by repeatedly linking the peptides.

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

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, myocardial infarction, heart failure or atrial fibrillation.

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

12. A composition comprising the peptide according to claim 3 as 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.

13. The composition according to claim 12, wherein The quasi-drug composition is provided in the form of an ointment, a patch, a powder, a gel, a spray, a tablet or a spray.

14. A composition as 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, comprising the peptide according to claim 3.

15. The composition according to claim 14, wherein The health functional food composition is used for preparing beverages, teas, spices, chewing gums or biscuits.

Images