Composition for prevention or treatment of oral diseases, containing SHLP2

Abstract

The present invention relates to a composition for the prevention or treatment of oral diseases, containing small humanin-like peptide 2 (SHLP2) as an active ingredient. The composition exhibits the effect of restoring tissue homeostasis in oral diseases including alveolar bone disease, such as alveolar bone damage, alveolar bone osteoporosis, alveolar bone osteomalacia, or alveolar bone osteopenia, and dental caries, gingivitis, periodontitis by reducing reactive oxygen species, inhibiting osteoblast apoptosis, increasing osteogenic differentiation factor expression, and improving mitochondrial function.

Description

SHLP2-containing composition for the prevention or treatment of oral diseases

[0001] The present invention relates to a new use of SHLP2 for the prevention or treatment of oral diseases.

[0002]

[0003] Oral disease refers to any condition that can occur within the oral cavity, including the teeth; representative examples include periodontal diseases such as dental caries, gingivitis, and periodontitis. Periodontal disease is an inflammatory condition that damages not only soft tissues surrounding the teeth, such as the periodontal ligament and gingiva, but also hard tissues like the alveolar bone. The alveolar bone is attached to the basal bone of the jawbone and serves to support the teeth. In the alveolar bone, bone formation by osteoblasts and bone resorption by osteoclasts proceed while maintaining a state of homeostasis; however, if bone resorption exceeds bone formation due to various causes, the alveolar bone lowers, exposing the tooth roots and eventually losing its function of supporting the teeth. This loss of alveolar bone is closely related to the progression of gingivitis and periodontitis and can further lead to alveolar bone diseases such as alveolar bone fracture, alveolar osteoporosis, alveolar osteomalacia, and alveolar osteopenia.

[0004] Oral and periodontal diseases are among the most common ailments affecting the adult population both domestically and internationally. They impose a significant burden on public health in terms of medical and social management costs, as well as a decline in individual quality of life. In fact, according to domestic statistics, the number of patients with periodontal disease has increased rapidly over the past few years, leading to the development of various surgical procedures and treatments targeting the condition. However, such surgical treatments entail significant psychological and economic burdens, and because they are often performed only after the disease has progressed to a certain stage, they limit early intervention and long-term management. Consequently, there has been a continuous demand for treatment and preventive measures capable of preventing periodontal and other oral diseases, improving symptoms, and enabling non-invasive, long-term management. Therefore, there is an urgent need to develop substances that can prevent these oral or periodontal diseases on an ongoing basis, alleviate symptoms, and even achieve therapeutic effects.

[0005] In particular, periodontitis is understood not merely as a condition limited to simple bacterial infection, but as a chronic inflammatory disease involving the complex interplay of systemic metabolic status, host immune responses, and aging-related cellular stress. Recent studies report that inflammatory aging—a chronic low-grade inflammatory state that accumulates with aging—significantly contributes to the structural breakdown of periodontal tissues and alveolar bone loss. In an environment of inflammatory aging, reactive oxygen species (ROS) are excessively generated; this leads to impaired intracellular mitochondrial function, induction of apoptosis, and disruption of the antioxidant system, thereby reducing the survival and differentiation functions of osteoblasts. This pathological condition results in delayed or insufficient alveolar bone regeneration even after microbiota reduction therapy.

[0006] To date, no composition has been proposed that utilizes SHLP2 as an active ingredient to inhibit oxidative stress and inflammaging and restore osteoblast function for oral diseases, particularly those where structural weakening of the alveolar bone and bone metabolic imbalance act as core pathologies, such as alveolar bone fracture, alveolar osteoporosis, alveolar osteomalacia, and alveolar osteopenia. Therefore, there is a need for a novel molecule-based therapeutic strategy capable of preventing or treating various oral and alveolar bone diseases by inhibiting degenerative changes in alveolar bone and periodontal tissues exacerbated by the accumulation of oxidative stress and aging, and by restoring the balance of bone remodeling.

[0007] [Prior Art Literature]

[0008] [Patent Literature]

[0009] (Patent Document 1) Korean Patent 10-2023-0132377

[0010]

[0011] Accordingly, the inventors have completed the present invention by confirming that SHLP2 (Small Humanin-Like Peptide 2) inhibits the functional decline of alveolar bone and periodontal tissues caused by oxidative stress and inflammatory aging, and restores the survival, differentiation, and mineralization functions of osteoblasts.

[0012]

[0013] Accordingly, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of oral diseases comprising SHLP2 as an active ingredient.

[0014] In addition, another objective of the present invention is to provide a food composition for preventing or improving oral diseases, comprising SHLP2 as an active ingredient.

[0015] In addition, another objective of the present invention is to provide a quasi-drug composition for preventing or improving oral diseases, comprising SHLP2 as an active ingredient.

[0016] In addition, another objective of the present invention is to provide a method for preventing or treating oral diseases, comprising the step of administering SHLP2 in a therapeutically effective amount to a subject who requires it.

[0017] In addition, another objective of the present invention is to provide a use of SHLP2 in the prevention or treatment of oral diseases.

[0018]

[0019] The present invention relates to a composition for the prevention or treatment of oral diseases comprising SHLP2 as an active ingredient, wherein the composition exhibits the effect of restoring tissue homeostasis in oral diseases including alveolar bone diseases such as alveolar bone fracture, alveolar osteoporosis, alveolar osteomalacia or alveolar osteopenia, dental caries, gingivitis, and periodontitis by reducing reactive oxygen species, inhibiting osteoblast apoptosis, increasing the expression of osteodifferentiation factors, and improving mitochondrial function.

[0020]

[0021] Figure 1 is a schematic diagram showing the mechanism of action of SHLP2, which restores damaged bone homeostasis and promotes the recovery of cell function by downregulating the apoptosis pathway induced by reactive oxygen species (ROS).

[0022] Figure 2 shows the viability and ROS analysis results of MC3T3-E1 cells treated with SHLP2 under H2O2-induced oxidative stress conditions [(A) Cell viability based on WST-1 analysis of cells exposed to 600 μM H2O2 for 24 hours after SHLP2 treatment for 1 day and (B) 3 days (n = 5; ** p < 0.01; *** p < 0.001). (C) Quantitative fluorescence intensity of intracellular ROS after 24 hours of exposure to 600 μM H2O2 (n = 3; ** p < 0.01; *** p < 0.001). (D) Representative fluorescence microscopy images showing ROS distribution in each group. Scale bar: 200 μm. Data are expressed as mean ± standard deviation (mean ± SD). Mock: Untreated osteoblasts cultured in growth medium. PC: H2O2-treated osteoblasts cultured in growth medium. 5 μM SHLP2: H2O2-treated osteoblasts cultured in growth medium containing 5 μM SHLP2. 10 μM SHLP2: H2O2-treated osteoblasts cultured in growth medium containing 10 μM SHLP2. WST-1: water-soluble tetrazolium-1].

[0023] Figure 3 shows the analysis of apoptosis-related gene expression in MC3T3-E1 cells subjected to oxidative stress [(A) Schematic diagram of oxidative stress response markers and the subsequent apoptotic signaling cascade (B) Relative expression levels of apoptosis-related genes measured on days 1 and 3 after treatment with 10 μM SHLP2 under H2O2-induced oxidative stress conditions (n ​​= 3; * p < 0.05; ** p < 0.01; *** p < 0.001). Data are expressed as mean ± standard deviation (mean ± SD). PC: H2O2-treated osteoblasts cultured in growth medium. 10 μM SHLP2: H2O2-treated osteoblasts cultured in growth medium containing 10 μM SHLP2].

[0024] Figure 4 shows the analysis of bone formation-related gene expression in MC3T3-E1 cells subjected to oxidative stress [(A) Relative expression levels of bone formation-related genes in MC3T3-E1 cells under H2O2-induced oxidative stress conditions after treatment with 10 μM SHLP2 for 3 and (B) 7 days (n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001). Data are expressed as mean ± standard deviation. PC: H2O2-treated osteoblasts cultured in osteodifferentiation medium. 10 μM SHLP2: H2O2-treated osteoblasts cultured in osteodifferentiation medium containing 10 μM SHLP2. ALP (alkaline phosphatase), RUNX2 (runt-related transcription factor 2), OSX (osterix), OCN (osteocalcin), BSP (bone sialoprotein), SPARC (secreted protein acidic and cysteine ​​rich)].

[0025] Figure 5 shows the alkaline phosphatase (ALP) staining analysis of oxidatively stressed MC3T3-E1 cells [(A) Protein-level ALP staining results in MC3T3-E1 cells under H2O2-induced oxidative stress conditions after treatment with 10 μM SHLP2 for 7 and (B) 14 days (n = 3; * p < 0.05; *** p < 0.001). Scale bar: 200 μm. Data are expressed as mean ± standard deviation. Mock: Untreated osteoblasts cultured in osteodifferentiation medium. PC: H2O2-treated osteoblasts cultured in osteodifferentiation medium. 10 μM SHLP2: H2O2-treated osteoblasts cultured in osteodifferentiation medium containing 10 μM SHLP2].

[0026] Figure 6 shows the results of biomineralization analysis of MC3T3-E1 cells subjected to oxidative stress [(A) Alizarin Red S staining results of protein levels in MC3T3-E1 cells under H2O2-induced oxidative stress conditions after treatment with 10 μM SHLP2 for 14 and (B) 21 days (n = 3; * p < 0.05; *** p < 0.001). Scale bar: 200 μm. Data are expressed as mean ± standard deviation. Mock: Untreated osteoblasts cultured in osteodifferentiation medium. PC: H2O2-treated osteoblasts cultured in osteodifferentiation medium. 10 μM SHLP2: H2O2-treated osteoblasts cultured in osteodifferentiation medium containing 10 μM SHLP2].

[0027]

[0028] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0029]

[0030] The present invention relates to a new use of SHLP2 for the prevention or treatment / improvement of oral diseases.

[0031] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of oral diseases, comprising SHLP2 as an active ingredient.

[0032] The above composition may include reducing reactive oxygen species (ROS), inhibiting apoptosis of pre-osteoblasts or osteoblasts, increasing the expression of at least one of RUNX2, ALP, BSP, and OSX, inhibiting the decline in mitochondrial function, improving mitochondrial metabolism, or reducing inflammatory responses in periodontal tissues.

[0033] Another aspect of the present invention provides a food composition for preventing or improving oral diseases, comprising SHLP2 as an active ingredient.

[0034] Another aspect of the present invention provides a quasi-drug composition for preventing or improving oral diseases, comprising SHLP2 as an active ingredient.

[0035] In the present invention, the term "SHLP2" is Small humanin-like peptide 2, which may be a peptide consisting of a total of 26 amino acids (Met-Gly-Val-Lys-Phe-Phe-Thr-Leu-Ser-Thr-Arg-Phe-Phe-Pro-Ser-Val-Gln-Arg-Ala-Val-Pro-Leu-Trp-Thr-Asn-Ser; SEQ ID NO: 1) or a peptide containing the amino acid sequence of SEQ ID NO: 1 or a variant thereof.

[0036] Furthermore, in the present invention, the term "oral disease" refers to any disease that may occur within the oral cavity, including the teeth. Specifically, it includes any disease that can be prevented, improved, and / or treated due to a decrease in the number of osteoclasts, an increase in the number of osteoblasts, a decrease in the volume of cancellous bone loss, an increase in the volume of alveolar bone, a decrease in the number of inflammatory cells, and an antimicrobial effect against harmful oral bacteria within the oral tissues. More specifically, the oral disease may be one or more diseases selected from the group consisting of alveolar bone breakage, alveolar bone osteoporosis, alveolar bone osteomalacia, alveolar bone osteopenia, dental caries, gingivitis, and periodontitis, but is not particularly limited thereto.

[0037] In addition, the term "improvement" as used in this invention refers to the effect of alleviating or reducing symptoms associated with oral or periodontal diseases by applying SHLP2.

[0038] The term "prevention" as used in this invention refers to any act of suppressing or delaying the onset of symptoms related to oral or periodontal disease by applying SHLP2 to subjects who do not yet show symptoms of oral or periodontal disease but have a high risk of developing such disease.

[0039] The term “treatment” as used in this invention refers to stopping or delaying the progression of a disease by applying SHLP2 to a subject exhibiting symptoms of oral or periodontal disease.

[0040] Since the composition of the present invention includes the SHLP2 (Small Humanin-Like Peptide 2) peptide, a variant thereof, a mixture with pharmaceutically acceptable excipients, etc., it can exhibit the same effects as reducing reactive oxygen species (ROS), inhibiting apoptosis of osteoblasts, increasing differentiation of osteoblasts, and restoring mitochondrial function in teeth or periodontal tissues in the oral cavity.

[0041] In the composition of the present invention, the SHLP2 peptide may be included in any amount (effective amount) depending on the specific use, formulation, and purpose of combination, as long as it can exhibit effects such as reducing reactive oxygen species (ROS), inhibiting apoptosis of osteoblasts, increasing differentiation and mineralization of osteoblasts, restoring mitochondrial function, reducing alveolar bone loss volume, increasing alveolar bone volume, and reducing the number of inflammatory cells in periodontal tissue. The “effective amount” refers to the amount of SHLP2 peptide that enables the composition of the present invention to exhibit pharmacological effects or physiological improvement effects to the subject during the period of administration by a medical professional, etc., and can typically be determined within the range of 0.0001% by weight to 50.0% by weight, more preferably 0.001% by weight to 30.0% by weight, based on the total weight of the composition. The selection of such an effective amount can be easily derived experimentally within the ordinary capacity of a person skilled in the art.

[0042] The subjects to which the composition of the present invention can be applied include mammals in general and humans, and it is particularly preferable to apply it to humans.

[0043] In addition, SHLP2 of the present invention can improve the function of alveolar bone degraded by oxidative stress and chronic inflammatory environments (inflammaging), thereby reducing the site of alveolar bone loss such as alveolar bone breakage, alveolar bone osteoporosis, alveolar bone osteomalacia, and alveolar bone osteopenia, and exhibiting the same effect of increasing alveolar bone volume and bone matrix calcification.

[0044] Furthermore, SHLP2 has been confirmed to reduce inflammatory responses within periodontal tissues, and through this, it can equally demonstrate the effect of inhibiting the progression of inflammatory oral diseases such as gingivitis and periodontitis.

[0045] As such, the SHLP2-containing composition of the present invention can be usefully applied to the prevention or improvement / treatment of various oral diseases through the inhibition of oxidative stress, recovery of mitochondrial metabolism, and increase in osteoblast function.

[0046] The pharmaceutical composition according to the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to SHLP2, and the adjuvants may include excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, lubricants, or flavoring agents.

[0047] The above pharmaceutical composition may preferably be formulated into a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

[0048] For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants, and colorants may also be included in the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose, or beta-lactose; corn sweeteners; natural and synthetic gums such as acacia, trackercanth, or sodium oleate; sodium stearate; magnesium stearate; sodium benzoate; sodium acetate; sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions may include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0049] The optimal values ​​for the individual dosage and administration interval of the pharmaceutical composition according to the present invention may be determined by considering the type and severity of oral or periodontal disease, the dosage form, the route of administration and site of application, and the age and health status of the subject. It is obvious to a person skilled in the art that such dosage and administration interval may be appropriately set by a medical professional based on ordinary clinical judgment.

[0050] The dosage described in this specification means a pharmaceutically acceptable amount sufficient to produce the intended therapeutic effect without causing excessive toxicity or side effects.

[0051] The administration of the pharmaceutical composition according to the present invention may be repeated at a sufficient frequency to achieve a therapeutic effect, and the dosage and frequency of administration may be adjusted or reduced as necessary according to normal clinical practice.

[0052] In addition, the pharmaceutical composition of the present invention may be administered through any conventional route of administration as long as the purpose of the present invention is achieved. For example, the pharmaceutical composition may be administered by intraperitoneal administration, intravenous administration, subcutaneous administration, intradermal administration, or oral administration, but is not limited thereto.

[0053] In the food composition of the present invention, the food may include a health functional food.

[0054] The term “health functional food” as used in this specification refers to a food manufactured or processed using raw materials or ingredients that have functional properties useful to the human body in accordance with the “Act on Health Functional Foods” (Article 3, Subparagraph 1), wherein “functional properties” means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions on the structure and function of the human body (Article 3, Subparagraph 2).

[0055] The health functional food of the present invention can be manufactured by methods commonly used in the field, and when manufacturing, it can be manufactured by adding raw materials and ingredients commonly used in the field.

[0056] In addition, the formulation of the above-mentioned health functional food may be applied without special restrictions as long as it is a formulation recognized as a health functional food.

[0057] The food composition of the present invention can be manufactured in various forms of formulations and, unlike general pharmaceuticals, has the advantage of having a low likelihood of side effects even when consumed over a long period of time as it is made from food. For example, the food composition may be one or more formulations selected from health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars, as well as beverages, gums, or candies, but is not limited thereto.

[0058] In the present invention, the food composition may be used alone as a functional food or added to various foods. Examples of such foods include beverages, vitamin complexes, and health supplements.

[0059] The food composition of the present invention may include ingredients commonly used in food manufacturing, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose, sucrose, and oligosaccharides; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents such as taumatin and stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) or synthetic flavoring agents such as saccharin and aspartame may be used.

[0060] In addition, the food composition of the present invention may further include food additives, and said food additives may be additives permitted under the "Food Additives Codex" unless otherwise specified. For example, chemically synthesized products, natural additives, or mixed preparations may be included, but are not limited thereto.

[0061] Foods containing the active ingredients of the present invention may include bread, rice cakes, confectionery, ice cream, dairy products, processed meat products, processed fish products, noodles, beverages, seasoning products, oil and fat products, and combinations thereof, and specific examples thereof may be appropriately selected by a person skilled in the art.

[0062] In addition, the food composition of the present invention may include vitamins, minerals, electrolytes, coloring agents, stabilizers, thickeners, pH adjusters, preservatives, carbonating agents, etc., and these components may be used alone or in combination.

[0063] Furthermore, since the quasi-drug composition of the present invention includes the SHLP2 peptide or a variant thereof, it can exhibit the same effects of reducing reactive oxygen species (ROS), inhibiting apoptosis of osteoblasts, increasing differentiation and mineralization of osteoblasts, reducing the volume of cancellous bone loss, increasing alveolar bone volume, and reducing the number of inflammatory cells in periodontal tissues, etc., in the oral cavity or periodontal tissues. In addition, the SHLP2 of the present invention can improve alveolar bone metabolism damaged by oxidative stress and impaired mitochondrial function, thereby exhibiting the same effects of inhibiting the progression of alveolar bone diseases such as alveolar bone fracture, alveolar osteoporosis, alveolar osteomalacia, and alveolar osteopenia, and promoting alveolar bone recovery. As such, the SHLP2-containing quasi-drug composition of the present invention can be usefully applied to the prevention or improvement of various oral diseases through the inhibition of oxidative damage to periodontal tissues, the restoration of mitochondrial metabolism, and the promotion of bone formation.

[0064] As used in this specification, the term “quasi-drug” means an article used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases of humans or animals, the effect of which is mild compared to that of pharmaceuticals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are articles excluding those used for pharmaceutical purposes, and may include articles used for the treatment or prevention of diseases of humans or animals, or articles that have a mild effect on the human body or do not act directly on it.

[0065] In one embodiment, the quasi-drug composition of the present invention may include an oral quasi-drug. In addition to the active ingredient, the quasi-drug composition may additionally include ingredients commonly used in oral quasi-drug compositions, such as, for example, abrasives, humectants, binders, foaming agents, sweeteners, preservatives, medicinal ingredients, flavoring agents, coloring agents, solvents, whitening agents, solubilizers, or pH adjusters, but is not limited thereto.

[0066] The quasi-drug composition of the present invention may be prepared in any formulation commonly manufactured in the field, and may be one or more formulations selected from, for example, toothpaste, mouthwash, gum, candy, oral spray, oral ointment, oral varnish, oral film, or gum massage cream, but is not particularly limited thereto.

[0067] In one embodiment, when the quasi-drug composition of the present invention is in the form of a toothpaste, the composition may include one or more of a humectant, an abrasive, a binder, a foaming agent, a flavoring agent, a sweetener, a coloring agent, a preservative, an active ingredient, a solvent, and a pH adjuster.

[0068]

[0069] In addition, the present invention includes a method for preventing or treating oral diseases, comprising the step of administering SHLP2 in a therapeutically effective amount to a subject who requires it.

[0070] Here, “therapeutically effective amount” means an amount sufficient to prevent the occurrence of oral disease or to alleviate, suppress, or improve symptoms.

[0071] The details regarding the administration form, administration route, method of use, and composition of the composition used therein for SHLP2 may be applied in the same way as the description provided for the composition containing SHLP2.

[0072] In addition, the present invention includes the use of SHLP2 in the prevention or treatment of oral diseases.

[0073] The form of provision, method of use, and method of application of SHLP2 may be applied in the same way as the descriptions regarding the composition and treatment method described in this specification.

[0074] According to an embodiment of the present invention, SHLP2 exhibits an effect of significantly reducing oxidative stress induced by reactive oxygen species (ROS). Oxidative stress is known to be a major factor causing tissue destruction and the exacerbation of inflammation in various oral diseases, including periodontal disease. By inhibiting the generation or accumulation of ROS, SHLP2 reduces oxidative damage at the cellular level and further contributes to stabilizing the microenvironment of periodontal tissues. Through this action, SHLP2 can effectively alleviate tissue degeneration and the inflammatory environment occurring in periodontal disease.

[0075] Furthermore, according to an embodiment of the present invention, SHLP2 promotes the survival of damaged cells by reducing apoptotic signals (p53, BAX, etc.) induced by oxidative stress and increasing anti-apoptotic signals (BCL-2, etc.). This apoptotic inhibitory effect allows periodontal tissue cells to maintain survival even in stressful environments, thereby minimizing tissue damage. In particular, SHLP2 has been confirmed to effectively inhibit the mitochondrial-mediated apoptotic pathway, thereby inhibiting the progression of cell function loss and tissue destruction commonly observed in periodontitis. Therefore, SHLP2 can function as a useful physiologically active factor for inhibiting the pathological progression of periodontal disease.

[0076] Furthermore, according to an embodiment of the present invention, SHLP2 exhibits the effect of restoring the expression of bone formation-related genes (RUNX2, OSX, ALP, OCN, etc.) that are suppressed by oxidative stress. Osteoblast function is essential for the maintenance and regeneration of alveolar bone, and in periodontitis, this function is significantly impaired by oxidative stress. Treatment with SHLP2 was confirmed to restore the physiological function of osteocytes to normal levels by increasing the expression of early and late bone differentiation markers. This implies that SHLP2 can effectively act to improve alveolar bone loss and reduced bone regeneration associated with periodontitis.

[0077] Furthermore, according to an embodiment of the present invention, SHLP2 increases alkaline phosphatase (ALP) activity and mineral deposition (Alizarin Red S-based mineralization analysis), thereby promoting the recovery of osteoblast function and alveolar bone formation. In the SHLP2-treated group, ALP activity and mineralization function, which were reduced by oxidative stress, were restored to levels approaching those of the normal group. This suggests that SHLP2 has an action that promotes the regeneration and recovery of alveolar bone tissue and can be effectively utilized to improve or prevent periodontitis and / or alveolar bone damage associated with periodontitis.

[0078] Figure 1 of the attached drawings is a schematic diagram illustrating that treatment with SHLP2 according to the present invention can contribute to the alleviation of tissue damage and the restoration of cell function in a periodontal tissue environment exposed to oxidative stress. The top of Figure 1 exemplifies a state in which increased reactive oxygen species, decreased cell function, and alveolar bone destruction occur due to periodontal disease, and the bottom exemplifies that cell survival and tissue regeneration can be improved following treatment with SHLP2. The present invention is not limited to a specific mechanism or pathway, and the effects of SHLP2 are supported by experimental results disclosed in the following examples.

[0079]

[0080]

[0081] [Example]

[0082] The present invention will be explained more specifically below by the following examples, but the present invention is not limited by the following examples.

[0083]

[0084] <Experimental Example>

[0085] The following experimental examples are intended to explain the experimental conditions and methods commonly applied to each embodiment according to the present invention.

[0086] 1. Cell culture

[0087] MC3T3-E1 pre-osteoblasts (mouse C57BL / 6 calvaria, subclone 4; ATCC) were cultured in low-glucose Dulbecco modified Eagle medium (DMEM, Cytiva, Marlborough, MA, USA) containing 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (PS, Cytiva) at 37°C under 5% CO₂ conditions. Hereinafter referred to as LG-DMEM in this experiment, this medium was selected to minimize experimental errors that could occur at excessive glucose concentrations, as it contains approximately 5 mM of glucose, which is similar to physiological concentrations. The medium was replaced every 3 days, and subcultures were performed in Trypsin / EDTA (Cytiva) when cell density was less than 80%.

[0088] The osteogenic differentiation medium was prepared by adding 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA) to the growth medium and was replaced every 2-3 days.

[0089]

[0090] 2. Establishment of an Oxidative Stress-Induced Cell Model

[0091] To construct a sub-toxic oxidative stress model related to cellular senescence and inflammaging as presented in a previous study [Han, D. et al. Cytoprotective effect of chlorogenic acid against hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells through PI3K / Akt-mediated Nrf2 / HO-1 signaling pathway. Oncotarget8, 14680 (2017)], 1 × 10⁴ cells were seeded into a 96-well plate and treated with H2O2 (200, 400, 600, 800 μM; 30% H2O2, Sigma Aldrich) for 24 hours. After treatment, the medium was removed, and cytotoxicity was evaluated by performing the WST-1 assay (EZ-Cytox, DoGenBio, Republic of Korea).

[0092] After adding 10% WST-1 to fresh medium and reacting at 37°C for 1 hour, the absorbance was measured at 450 nm (BioTek, Winooski, VT, USA). The concentration at which cell viability reached approximately 70% was set as the subtoxic oxidative stress limit.

[0093]

[0094] 3. Evaluation of SHLP2 cytotoxicity in osteoblasts

[0095] SHLP2 was synthesized by AnyGen (Gwangju, Republic of Korea) in a previous study [Kim, SK et al. Mitochondria-derived peptide SHLP2 regulates energy homeostasis through the activation of hypothalamic neurons. Nat Commun 14, 4321 (2023)]. A 1 mM stock solution was prepared by dissolving 1 mg of SHLP2 in 330 μL of sterile distilled water (Chong Kun Dang Pharmaceutical Co., Ltd., Republic of Korea). SHLP2 at concentrations of 5 μM, 10 μM, and 20 μM were prepared by diluting the 1 mM stock solution in the medium, and cytotoxicity was evaluated using the same WST-1 assay after treating MC3T3-E1 cells for 24 hours.

[0096]

[0097] 4. Analysis of Mitochondrial Activity

[0098] The WST-1 assay was performed to evaluate cellular metabolic activity (=mitochondrial function). Cells were seeded at 2 × 10⁴ per 24-well plate and cultured for 24 hours (37 ℃, 5% CO2). Subsequently, oxidative stress was induced for 24 hours by replacing the growth medium with fresh medium containing 600 μM H2O2. After H2O2 treatment, DPBS (Cytiva, Ca 2+ / Mg 2+ Washed twice with (no addition) and cultured in growth medium containing SHLP2 for 1, 3, and 7 days. At each time point, 10% (v / v) of WST-1 solution was added and reacted at 37°C for 1 hour, after which the supernatant was transferred to a 96-well plate and the absorbance was measured at 450 nm.

[0099]

[0100] 5. Reactive Oxygen Species (ROS) Analysis

[0101] Intracellular ROS was analyzed using CellROX™Green (ThermoFisher Scientific).

[0102] Cells were seeded at 5 × 10⁴ per 24-well plate and cultured for 24 hours. Subsequently, the growth medium was replaced with a medium containing 600 μM H2O2 to induce oxidative stress for 24 hours. After removing H2O2, the cells were washed twice with DPBS and cultured for 24 hours in a growth medium containing SHLP2. For fluorescence analysis, 5 μM CellROX working solution was applied for 30 minutes, followed by washing with DPBS and fixation with 3.7% paraformaldehyde. Fluorescence images were acquired using a fluorescence microscope (Olympus IX73; ×10), and fluorescence intensity was quantified using Zen software (version 3.4, Carl Zeiss).

[0103]

[0104] 6. Quantitative PCR Analysis

[0105] A cell is 1 × 10⁶ 5 Samplings were dispensed into 12-well plates and cultured for 24 hours. Afterward, oxidative stress was induced with H2O2 (600 μM) for 24 hours, followed by washing twice with DPBS.

[0106] 1) Analysis of apoptosis genes

[0107] After inducing oxidative stress, cells were cultured in growth medium containing SHLP2 for 1 and 3 days. Cells cultured in growth medium after H2O2 treatment were used as a positive control (PC).

[0108] 2) Bone formation gene analysis

[0109] Cells were cultured for 3 and 7 days in osteogenic differentiation medium containing SHLP2. Cells cultured in osteogenic differentiation medium after H2O2 treatment were set as PCs. Total RNA was extracted with Qiazol (Qiagen), and cDNA was synthesized using the PrimeScript™RT kit (Takara Bio). qPCR was performed using SYBR®Premix Ex Taq™II (Takara Bio), and primer information is presented in Table 1 below. Expression levels were 2 -ΔΔCT It was analyzed using the method and GAPDH was used as an intrinsic control.

[0110] [Table 1]

[0111]

[0112]

[0113]

[0114] 7. ALP (Alkaline phosphatase) staining

[0115] MC3T3-E1 cells 1 × 10 5 Cells were seeded into 24-well plates. After 24 hours, 24-hour oxidative stress was induced with H2O2 (600 μM), and after washing, the cells were cultured for 7 and 14 days in growth medium containing SHLP2. Cells without oxidative stress cultured in osteogenic differentiation medium (mock) were designated as the negative control (NC), and cells cultured in H2O2+ osteogenic differentiation medium were designated as the positive control (PC). At each time point, cells were fixed with 4% paraformaldehyde and stained with SIGMAFAST™BCIP® / NBT (Sigma Aldrich) at 25°C for 1 hour. Cells stained purple were photographed using a light microscope (×10), and the stained area was quantified using ImageJ.

[0116]

[0117] 8. Mineralization Analysis

[0118] MC3T3-E1 cells were cultured in the same manner as in the ALP experiment described above, and then cultured for 14 and 21 days in a growth medium containing SHLP2. The cells were fixed with 4% paraformaldehyde and stained with ARS (Alizarin Red S) solution. Red mineral nodules were observed under a light microscope (×10). For quantification, ARS was dissolved in 10 mM sodium phosphate (pH 7.0) and 10% cetylpyridinium chloride solution, and the absorbance was measured at 562 nm.

[0119]

[0120] 9. Statistical Analysis

[0121] Statistical analysis was performed using SPSS v26 (IBM, Armonk, NY, USA). All experiments were repeated at least three times, and each condition was measured at least three times. Data were presented as mean ± standard deviation.

[0122] One-way ANOVA and Tukey's post-hoc test were used for comparisons between multiple groups. T-tests were used for comparisons between two groups. A p value < 0.05 was considered statistically significant.

[0123]

[0124] Example 1: Effects of SHLP2 Treatment on Cell Viability and ROS Activity in Oxidatively Stressed Cells

[0125] The sub-toxic oxidative stress threshold of pre-osteoblasts was determined through cell viability analysis using various concentrations of H2O2. Treatment with 600 μM H2O2 resulted in a cell viability of approximately 71%, and accordingly, this concentration was selected as the sub-toxic oxidative stress condition for subsequent experiments. Screening of SHLP2 at concentrations of 5 μM, 10 μM, and 20 μM showed that cell viability increased up to 10 μM, while at 20 μM, it was similar to that of the untreated group. Based on these results, the 600 μM H2O2 condition was established as the sub-toxic oxidative stress environment for subsequent experiments.

[0126] Analysis of cell metabolic activity showed no significant difference at 24 hours after SHLP2 treatment (Fig. 2A). However, after 3 days, the metabolic activity of the 10 μM SHLP2-treated group increased by 11.6% compared to PC (positive control) (p < 0.001; Fig. 2B). Immunofluorescence analysis showed a significant decrease in ROS levels after 10 μM SHLP2 treatment (p < 0.001; Figs. 2C-D).

[0127] In summary, 10 μM SHLP2 effectively inhibited ROS production along with distinct cell proliferation activity. Therefore, 10 μM SHLP2 was selected as the condition for comparison with the PC group in subsequent analyses of anti-apoptotic and osteogenic activity.

[0128]

[0129] Example 2: Effects of SHLP2 on Oxidative Stress and Apoptosis Signaling

[0130] To investigate the inhibition of ROS-induced apoptosis, mRNA expression of oxidative stress response and apoptosis-related genes was analyzed (Fig. 3A). On day 1 of SHLP2 treatment, the expression of SOD1, an initial response marker, significantly increased (p < 0.001), and SOD2 expression also showed an increase. Major components of the apoptosis signaling pathway decreased distinctly, with p53 expression decreasing significantly (p < 0.01). The anti-apoptotic gene BCL-2 increased upon SHLP2 treatment but was not statistically significant (Fig. 3B).

[0131] On day 3, the SHLP2-treated group showed significantly lower expression changes for both oxidative stress and apoptosis markers, which means that SHLP2 increases cell resistance in a subtoxic oxidative stress environment (Fig. 3B, Table 2).

[0132] [Table 2]

[0133] p-values ​​of in vitro qPCR analysis for oxidative stress and apoptosis signaling markers

[0134]

[0135]

[0136] Example 3: Increase in osteogenic activity following SHLP2 treatment

[0137] The functional recovery of pre-osteoblasts was evaluated by comparing the expression of bone formation markers after 3 and 7 days of culture. On day 3 of SHLP2 treatment, OSX and RUNX2 showed the most distinct increase among the major bone formation markers. In addition, BSP, ALP, SPARC, and OCN all increased significantly (Fig. 4A). On day 7, the SHLP2-treated group also showed a statistically significant increase in all markers, and a multi-fold increase in expression was confirmed, particularly in ALP, BSP, and OSX (Fig. 4B).

[0138] In addition, the increase in ALP expression on day 7 after SHLP2 treatment was also confirmed by ALP staining (Fig. 5A). Osteoblast activity in the SHLP2-treated group recovered to 85.6% of the mock group's level, while the PC group was only 50%. Furthermore, after 14 days, ALP activity in SHLP2-treated cells reached 95.6% of the mock group's level, but the PC group was at 61.8% (Fig. 5B).

[0139] These results show that 10 μM SHLP2 induces a distinct increase in bone formation marker expression under oxidative stress and demonstrates the ability of MC3T3-E1 cells to recover function.

[0140]

[0141] Example 4: Effect of SHLP2 on Increased Mineralization of Pre-osteoblasts

[0142] Late-stage osteodifferentiation was confirmed through ARS mineralization analysis. On day 14, the SHLP2-treated group showed osteoblast activity at 94.92% of the mock group, while the PC group was only 68.45% (Fig. 6A). On day 21, the ARS staining intensity increased in both the PC group and the SHLP2-treated group, confirming that mineral deposition increased over time. However, compared to the mock group, the PC group showed a low level of 72.1%, whereas the SHLP2-treated group maintained a high response of 94.9% (Fig. 6B).

[0143]

[0144] In summary, 10 μM SHLP2 treatment effectively reversed oxidative stress in osteoblasts, inhibited apoptosis, and maintained osteogenic activity. These results demonstrate that SHLP2 has potential as a strategy to improve or treat general oral diseases, including periodontal disease, by improving the tissue microenvironment in the context of periodontitis or inflammatory aging.

Claims

1. A food composition for preventing or improving oral diseases, comprising SHLP2 (Small Humanin-Like Peptide 2) as an active ingredient.

2. In Paragraph 1, A pharmaceutical composition in which the above SHLP2 is a peptide or a variant thereof comprising the amino acid sequence of SEQ ID NO:

1.

3. In Paragraph 1, A pharmaceutical composition that reduces reactive oxygen species (ROS).

4. In Paragraph 1, A pharmaceutical composition that inhibits apoptosis of pre-osteoblasts or osteoblasts.

5. In Paragraph 1, A pharmaceutical composition that increases the expression of at least one osteodifferentiation-related factor among RUNX2, ALP, BSP, and OSX.

6. In Paragraph 1, A pharmaceutical composition that inhibits the decline of mitochondrial function or improves mitochondrial metabolism.

7. In Paragraph 1, A pharmaceutical composition that reduces inflammatory responses in periodontal tissues.

8. In Paragraph 1, A pharmaceutical composition wherein the above oral disease is one or more selected from the group consisting of alveolar bone breakage, alveolar bone osteoporosis, alveolar bone osteomalacia, alveolar bone osteopenia, dental caries, gingivitis, and periodontitis.

9. A food composition for preventing or improving oral diseases, comprising SHLP2 (Small Humanin-Like Peptide 2) as an active ingredient.

10. In Paragraph 9, The above food is a food composition that is a health functional food.

11. A quasi-drug composition for the prevention or improvement of oral diseases, comprising SHLP2 (Small Humanin-Like Peptide 2) as an active ingredient.

12. A method for preventing or treating oral disease, comprising the step of administering SHLP2 (Small Humanin-Like Peptide 2) in a therapeutically effective amount to a subject who requires it.

13. Use of SHLP2 (Small Humanin-Like Peptide 2) in the prevention or treatment of oral diseases.

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