Method for removing advanced glycation end product in tissue, and method for inhibiting aging using same

Abstract

The present invention relates to a method for removing intracellular advanced glycation end products, and a method for inhibiting aging using same. Advanced glycation end products accumulate in tissues in the body and exert adverse effects on various tissues of the body. At present, a reduction in the intake of advanced glycation end products through dietary control is considered almost the only available approach to reducing their accumulation. However, the method for removing intracellular advanced glycation end products of the present invention not only inhibits the production of advanced glycation end products, but also effectively promotes the degradation of already-formed advanced glycation end products, and thus is expected to be applicable to the prevention, alleviation, or treatment of aging-related chronic diseases.

Description

Method for removing advanced glycation end products in tissues and method for inhibiting aging using the same

[0001] The present invention relates to a method for removing advanced glycation end products in a tissue and a method for inhibiting aging using the same.

[0002] Sugar molecules react nonenzymatically with amino acid residues in biomolecules, particularly proteins, inducing their denaturation. This biochemical reaction is called glycation. This denaturation can cause the protein to lose its original function or perform abnormal functions. Glycation proceeds through a series of reactions to form chemically very stable products, by-products called advanced glycation end-products (AGEs). Over time, these AGEs accumulate in body tissues, causing adverse effects on various tissues. Furthermore, high-temperature food processing can generate large amounts of AGEs, and dietary intake of AGEs also significantly contributes to their accumulation in the body.

[0003] To date, numerous studies have supported the direct link between the accumulation of advanced glycation end products (AGEs) and various health problems. For example, AGEs can cause and accelerate chronic diseases such as diabetic complications, cardiovascular disease, and Alzheimer's disease by reducing vascular elasticity, inducing inflammation, and disrupting intercellular signaling. Furthermore, they contribute to the development of age-related diseases by accelerating the aging process through tissue stiffening, decreased elasticity, and increased inflammation. Therefore, numerous attempts and efforts have been made recently to develop compounds or drugs that can inhibit the formation of AGEs to treat and prevent chronic diseases, including diabetic complications, and to delay and improve aging.

[0004] Conventional techniques targeting AGEs primarily focus on inhibiting AGE formation or reducing their accumulation in the early stages, utilizing specific compounds or drugs. This means that existing techniques have clear limitations in effectively removing AGEs already accumulated in tissues. Furthermore, most current techniques are often ineffective, have side effects, or are difficult to apply in clinical settings. Therefore, reducing AGE intake through dietary interventions appears to be the only viable method for reducing their accumulation.

[0005] Accordingly, the present invention was conceived to address the above problems and relates to a method for effectively removing advanced glycation end products (AGEs) within tissues. Using the method of the present invention, it is possible not only to inhibit the formation of AGEs but also to promote the degradation of already formed AGEs. The method for removing AGEs of the present invention can ultimately be used to prevent, improve, or treat age-related chronic diseases, and is therefore expected to find significant application in the medical and cosmetic fields.

[0006] One object of the present invention is to provide a method for removing advanced glycation end-products in cells or tissues.

[0007] Another object of the present invention is to provide a device for preventing or improving skin aging by using the method for removing the advanced glycation end products.

[0008] Another object of the present invention is to provide a method for preventing or improving skin aging using the method for removing the advanced glycation end products.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0011] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0012] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0013] Sugar molecules react nonenzymatically with amino acid residues in biomolecules, particularly proteins, inducing their denaturation. This biochemical reaction is called glycation. This denaturation can cause the protein to lose its original function or perform abnormal functions. Glycation proceeds through a series of reactions to form chemically very stable products, known as advanced glycation end-products (AGEs). Over time, these AGEs accumulate in body tissues, causing adverse effects on various tissues.

[0014] In one aspect of the present invention, the present invention provides a method for removing advanced glycation end-products, comprising the step of injecting wave energy into a cell or tissue.

[0015] In the method for removing advanced glycation end products of the present invention, the wave energy injected into the cell or tissue may be expressed as an ultrasonic wave having a frequency of 20,000 to 50,000 Hz or a high frequency having a frequency of 50,000 Hz or more, and preferably, it is energy having an input voltage of 47.4 Vpp to 71.1 Vpp, or 155.68 mW / cm 2 1,660 mW / cm 2 It may be, but is not limited to, energy of, or energy injected at a 100% duty cycle.

[0016] In the method for removing the final glycation product of the present invention, any cell or tissue that constitutes a living body may be used, but preferably, it may be a skin cell or skin tissue, and most preferably, it may be skin tissue.

[0017] In the method for removing advanced glycation end products of the present invention, the advanced glycation end product is not limited as long as it is a sugar molecule that is denatured (glycated) by non-enzymatic reaction with an amino acid residue of a biomolecule, particularly a protein, but preferably, it is Fibronectin, Heparan Sulfate Proteoglycan, Elastin microfibril interface-located protein 1 (EMILIN-1), Fibulin-2, Tenascin-C, Collagen alpha-1(XII) chain, Collagen alpha-2(VI) chain, Collagen alpha-1(I) chain, Fibrillin-1, Annexin A2, Collagen alpha-1(I) chain, It may be at least one selected from the group consisting of collagen alpha-1(VI) chain, transglutaminase-2, elastin microfibril interface-located protein 2 (EMILIN-2), and collagen alpha-1(XVI) chain.

[0018] In the method for removing advanced glycation end products of the present invention, the removal of advanced glycation end products may be suppression of the production of advanced glycation end products or promotion of the decomposition of advanced glycation end products already produced, and may include both suppression of the production of advanced glycation end products and promotion of the decomposition of advanced glycation end products already produced.

[0019] In another aspect of the present invention, the present invention provides a device for preventing or improving cell or tissue aging, comprising an injection unit that injects wave energy into a cell or tissue.

[0020] In the above-described device for preventing or improving aging of the present invention, the wave energy injected into the cell or tissue may be expressed as an ultrasound having a frequency of 20,000 to 50,000 Hz or a high frequency having a frequency of 50,000 Hz or more, and preferably, it is energy having an input voltage of 47.4 Vpp to 71.1 Vpp, or 155.68 mW / cm 2 1,660 mW / cm 2 It may be, but is not limited to, energy of, or energy injected at a 100% duty cycle.

[0021] In the device for preventing or improving aging of the present invention, any cell or tissue that constitutes a living body may be used, but preferably, it may be a skin cell or skin tissue, and most preferably, it may be skin tissue. When the device of the present invention is applied to skin tissue, the skin aging to be prevented or improved may be skin oxidation, skin dryness, skin inflammation, skin pigmentation, decreased skin elasticity, or skin wrinkle formation.

[0022] The above-described device for preventing or improving aging of the present invention can also be used as a device for preventing or treating aging diseases. At this time, the aging disease that is the target of prevention or treatment may be at least one selected from the group consisting of diabetes, diabetic complications, hyperlipidemia, hyperglycemia, cardiovascular disease, degenerative brain disease, autism spectrum disorder, arteriosclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, skin fibrosis, pulmonary fibrosis, renal fibrosis, and cardiac fibrosis, and the diabetes may be type 2 diabetes, and the diabetic complications may be at least one selected from the group consisting of diabetic nephropathy, diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic foot ulcer, diabetic cardiovascular disease, diabetic arteriosclerosis, diabetic osteoporosis, diabetic sarcopenia, and obesity, and the degenerative brain disease may be at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, Lou Gehrig's disease, spinocerebellar degeneration, Friedreich's ataxia, spinocerebellar ataxia, It may be one or more selected from the group consisting of Machado-Joseph disease, dystonia, progressive supranuclear palsy, cognitive impairment, senile dementia, dementia with Lewy bodies, frontotemporal dementia, vascular dementia, alcoholic dementia, early-onset dementia, temporal lobe epilepsy, and stroke.

[0023] In another aspect of the present invention, the present invention provides a method for preventing or improving cell or tissue aging, comprising the step of injecting wave energy into a cell or tissue.

[0024] In the method for preventing or improving aging of the present invention, the wave energy injected into the cell or tissue may be expressed as an ultrasound having a frequency of 20,000 to 50,000 Hz or a high frequency having a frequency of 50,000 Hz or more, and preferably, it is energy having an input voltage of 47.4 Vpp to 71.1 Vpp, or 155.68 mW / cm 2 1,660 mW / cm 2It may be, but is not limited to, energy of, or energy injected at a 100% duty cycle.

[0025] In the method for preventing or improving aging of the present invention, any cell or tissue that constitutes a living body may be used, but preferably, it may be a skin cell or skin tissue, and most preferably, it may be skin tissue. When the device of the present invention is applied to skin tissue, the skin aging to be prevented or improved may be skin oxidation, skin dryness, skin inflammation, skin pigmentation, decreased skin elasticity, or skin wrinkle formation.

[0026] The above method for preventing or improving aging of the present invention can also be used as a method for preventing or treating aging diseases. At this time, the aging disease that is the target of prevention or treatment may be at least one selected from the group consisting of diabetes, diabetic complications, hyperlipidemia, hyperglycemia, cardiovascular disease, degenerative brain disease, autism spectrum disorder, arteriosclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, skin fibrosis, pulmonary fibrosis, renal fibrosis, and cardiac fibrosis, and the diabetes may be type 2 diabetes, and the diabetic complications may be at least one selected from the group consisting of diabetic nephropathy, diabetic retinopathy, diabetic cataract, diabetic neuropathy, diabetic foot ulcer, diabetic cardiovascular disease, diabetic arteriosclerosis, diabetic osteoporosis, diabetic sarcopenia, and obesity, and the degenerative brain disease may be at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, Lou Gehrig's disease, spinocerebellar degeneration, Friedreich's ataxia, spinocerebellar ataxia, It may be one or more selected from the group consisting of Machado-Joseph disease, dystonia, progressive supranuclear palsy, cognitive impairment, senile dementia, dementia with Lewy bodies, frontotemporal dementia, vascular dementia, alcoholic dementia, early-onset dementia, temporal lobe epilepsy, and stroke.

[0027] Hereinafter, the present invention will be described in detail based on examples.

[0028] The method for removing advanced glycation end products within a cell of the present invention is expected to be used for the prevention, improvement, or treatment of chronic diseases related to aging, as it not only suppresses the production of advanced glycation end products but also has an excellent effect of promoting the decomposition of already produced advanced glycation end products.

[0029] FIG. 1 is a result of measuring SATA (spatial average-temporal average) intensity according to input voltage and duty cycle when applying ultrasound / high frequency treatment to a multilayer structure in vitro aging model using an ultrasound / high frequency application device manufactured in the present invention in one embodiment of the present invention.

[0030] Figure 2 is a schematic diagram showing an experimental process for confirming the effect of ultrasound / high frequency on inhibition and delay of advanced glycation end product formation in one embodiment of the present invention.

[0031] Figure 3 is a result of confirming the differential effect on inhibition of advanced glycation end product formation according to the SATA intensity of ultrasonic / high frequency in one embodiment of the present invention.

[0032] FIG. 4 is a schematic diagram showing an experimental process for confirming the effect of ultrasound / high frequency on advanced glycation end products already accumulated in ECM in one embodiment of the present invention.

[0033] Figure 5 is a result of confirming the differential effect on removal of advanced glycation end products according to SATA intensity of ultrasonic / high frequency in one embodiment of the present invention.

[0034] FIG. 6 is a schematic diagram showing an experimental process for comparing advanced glycation end products in an in vitro tissue model before and after ultrasound / radiofrequency treatment using mass spectrometry in one embodiment of the present invention.

[0035] Figure 7 shows the results of confirming the effect of ultrasound / radio frequency on removing advanced glycation end products in the top 15 proteins that underwent glycation in an in vitro aging tissue model in one embodiment of the present invention.

[0036] Figure 8 is a result of confirming the effect of ultrasound / high frequency on removing advanced glycation end products for each protein in an in vitro aging tissue model according to one embodiment of the present invention.

[0037] FIG. 9 is a result of confirming the distribution of advanced glycation end products existing in type 1 collagen α1 chain / α2 chain or fibronectin in an in vitro aging tissue model before and after ultrasound / radiofrequency treatment in one embodiment of the present invention.

[0038] FIG. 10 is a result of confirming lysine and arginine residues in type 1 collagen α1 chain / α2 chain or fibronectin with advanced glycation end products removed in an in vitro aging tissue model after ultrasound / radiofrequency treatment in one embodiment of the present invention.

[0039] Figure 11 shows the results of confirming the effect of removing advanced glycation end products according to the intensity of ultrasound / high frequency in aged mouse skin tissue in one embodiment of the present invention.

[0040] Figure 12 shows the results of confirming the effect of removing advanced glycation end products according to the ultrasound / radiofrequency treatment time in aged mouse skin tissue in one embodiment of the present invention.

[0041] Figure 13 shows the results of confirming the effect of removing advanced glycation end products in skin tissue according to mouse age in one embodiment of the present invention.

[0042] To confirm the effect of ultrasound / radiofrequency on the inhibition and delay of advanced glycation end product (AGE) formation, the SATA intensity of ultrasound delivered to an in vitro aged tissue model was controlled through the input voltage, and the differential effects were investigated. To this end, the control group was induced to glycate the in vitro tissue model through reaction with 500 mM ribose for one week without ultrasound treatment, and the experimental group was treated with ultrasound for 1 hour per day for a total of 7 days at a 100% duty cycle during the reaction with ribose. A schematic diagram of the experimental process is shown in Fig. 2. As a result, the group with an input voltage of 31.6 Vpp showed no significant effect, but the group with 47.4 Vpp showed a significant effect. This effect reached its maximum effect at 71.1 Vpp. This suggests that there is an effective range of ultrasound / radiofrequency intensity that is effective in inhibiting and delaying AGE formation. As a result, it was observed that the formation of advanced glycation end products was inhibited by up to about 87% compared to before ultrasound / high frequency treatment, and about 155.68 mW / cm 2 It was confirmed to have a threshold of (47.4 Vpp).

[0043] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0044] Example

[0045] [Experimental Method]

[0046] 1. Ultrasound stimulation experiment in an aging mouse model

[0047] All aging mouse models used in the experiments were natural aging models, consisting of mice aged 18-20 months. Mice were anesthetized with an isoflurane vaporizer and stabilized for stimulation. All hair on the dorsal skin, the site of ultrasound stimulation, was removed with a depilatory agent, and the area was washed with alcohol swabs and phosphate-buffered saline (PBS) to prevent secondary infection. Because mouse skin is very thin and stretchy, the skin around the stimulation site was secured with medical tape. The ultrasound stimulation site was marked, and the ultrasound transducer was fixed 5 mm above. An acoustic matching layer, hydrogel, was applied between the ultrasound transducer and the mouse skin without air bubbles to ensure that the ultrasound generated by the transducer was transmitted to the skin without loss.

[0048] Ultrasound stimulation was performed at an intensity that ensured a mechanical index of 1.9 or less, in accordance with US safety guidelines. Stimulation was performed at a frequency of 4.7 MHz for 1 hour daily for 5 days, with the total anesthesia time not exceeding 3 hours.

[0049] After completing the experiment, the mice were anesthetized with 4% isoflurane and euthanized in a 100% chamber. Afterwards, the dorsal skin tissues from the ultrasound-stimulated and non-stimulated areas were collected using tweezers and medical scissors. The collected skin tissue samples were cut into 2 mm x 2 mm pieces and fixed with 1 ml of 4% paraformaldehyde at room temperature for 12 hours. The fixed skin tissues were washed with PBS and then paraffin-embedded. The embedded skin tissue samples were then sectioned at 5 ㎛ thickness and placed on slide glass.

[0050] 2. Immunostaining

[0051] After washing the samples twice with Dulbecco's phosphate-buffered saline (DPBS), DBPS was completely removed, and the samples were fixed with a 4% paraformaldehyde solution at room temperature for 20 minutes. After fixation, the paraformaldehyde solution was completely removed, and the samples were washed three times with DPBS. After washing, the DPBS was completely removed, and the samples were permeabilized by infiltration with a 0.15% (v / v) Triton X-100 solution diluted in DBPS for 15 minutes at room temperature. After infiltration, the Triton X-100 solution was completely removed, and the samples were washed three times with DPBS. After washing, the DPBS was completely removed, and to prevent nonspecific binding, the samples were treated with a 1% (w / v) bovine serum albumin (BSA) solution diluted in DPBS for 1 hour at room temperature. After blocking, the BSA solution was completely removed, and the samples were washed three times with DPBS.

[0052] After washing, the DPBS was completely removed, and the primary antibody was diluted in a 1% (w / v) BSA solution at the concentration recommended by the manufacturer for each antibody and reacted with the sample at 4°C for 16-24 hours. The sample was then washed three times with DPBS, the washed DPBS was completely removed, and the secondary antibody was diluted in a 1% (w / v) BSA solution at the concentration recommended by the manufacturer and reacted with the sample at room temperature for 1 hour 30 minutes to 2 hours. After washing the sample three times with DPBS, the washed DPBS was completely removed, and phalloidin was reacted with the sample at a concentration of 5 μg / ㎖ for 45 minutes at room temperature. After the reacted phalloidin was completely removed, the sample was washed three times with DPBS, the DPBS was completely removed, and DAPI was reacted with the sample at a concentration of 4 μg / ㎖ for 15 minutes at room temperature. The reacted DAPI was completely removed, and the sample was washed three times with DPBS. All subsequent steps after secondary antibody treatment were performed in a dark, light-shielded environment. A few drops of mounting solution were placed on the slide glass and pressed against the cover glass with the sample. After completely drying the sample at room temperature, it was stored at -20°C or -80°C.

[0053] 3. Proteomic mass analysis of multilayer cell-derived extracellular matrix

[0054] A multilayer in vitro aging model was fabricated using the method described in our previous study (Korean Patent Application No. 10-2023-0068882). Specifically, a glass cover glass was treated with oxygen plasma at 30 W for 1 minute to temporarily transform its hydrophobic surface into a hydrophilic one. This was then subjected to 37 o After coating with fibronectin for more than 4 hours in C, placed in a 35pi petri dish, 8x10 4 / cm 2NIH / 3T3 cells were seeded. The cells were cultured to form 100% confluency on the cover glass surface, and the cellular component (CC) was removed by treatment with an extraction buffer containing 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton X-100, and 0.001% poly-L-lysine (PLL), and only the highly pure extracellular matrix (ECM) was extracted. The cells were washed three or more times with Dulbecco's phosphate buffer saline (DPBS), and the cell culture was re-performed on the ECM monolayer. When the cells formed 100% confluency, the ECM was re-extracted. By repeating the cell culture and ECM extraction steps, a multilayered in vitro aging model was manufactured. Finally, the produced multilayered ECM was incubated in 500 mM ribose dissolved in DPBS for 37 min. o A glycated model was created by reacting for 3 weeks while maintaining the C condition.

[0055] Afterwards, the cell-derived extracellular matrix was harvested from the cover glass using a cell scraper, and the sample was sufficiently dissolved using a buffer consisting of 5% (w / v) sodium dodecyl sulfate (SDS), 50 mM Triethylammonium bicarbonate (TEAB), pH 8.5. To primarily filter out impurities, the sample was centrifuged at 13,000 rpm for 8 minutes at room temperature, the supernatant was collected, and the protein in the sample was quantified using the bicinchoninic acid (BCA) method, of which 300 μg of protein was used. Tris(2-carboxyethyl)phosphine (TCEP) diluted in distilled water was added to the sample to a final concentration of 5 mM, and the sample was reduced by treating it at room temperature for 10 minutes. Iodoacetamide (IAA) diluted in isopropyl alcohol was added to the sample to a final concentration of 20 mM and treated at room temperature for 10 minutes to alkylate the sample. Phosphoric acid diluted in distilled water was added to the sample to a final concentration of 1.1% and mixed well.

[0056] To capture the protein, a binding / wash buffer consisting of a 100 mM TEAB solution diluted in 90% methanol was prepared, and the mixture was mixed well with 300 μg of the previously transferred protein. The mixture was placed on an S-Trap column in a tube of an appropriate volume and centrifuged at 4,000 g for 30 seconds to capture the protein. Afterwards, centrifugation was performed at 4,000 g for 30 seconds with the S-Trap column using the binding / wash buffer, and the buffer that passed through the column was discarded. This process was repeated three times. Finally, centrifugation was performed once more at 4,000 g for 1 minute with the S-Trap and the column was transferred to a new tube. A sufficient amount of trypsin was dissolved in a 50 mM TEAB solution to make a digestion buffer, which was placed on the column and incubated at 47°C for 1-2 hours or at 37°C for 16 hours. In this study, 30 μg of trypsin was completely dissolved in 125 μl of 50 mM TEAB solution to make a digestion buffer. After the reaction, a 50 mM TEAB solution diluted in distilled water was added and centrifuged once at 4,000 rcf for 1 minute. After the first centrifugation, an equal amount of a 0.2% formic acid solution diluted in distilled water was added and centrifuged again at 4,000 rcf for 1 minute. After this, an equal amount of a 50% acetonitrile solution diluted in distilled water was added and centrifuged a third time at 4,000 rcf for 1 minute to recover hydrophobic peptides. After centrifugation, the eluted peptides were dried and resuspended in a 0.1% formic acid solution diluted in distilled water for mass spectrometry.

[0057] Liquid chromatography conditions (ion pair reversal method, separation column type, sample injection volume, flow rate, etc.) for peptide separation were set and a NanoLC system was used. Here, peptides were separated through reversed-phase-high performance liquid chromatography (RP-HPLC) on a 75 ㎛ x 50 cm analytical column. MS conditions (electrospray ionization, collision energy, mass range) were set, and 500 ng of peptides were injected. They were ionized and data were acquired through data-dependent acquisition (DDA) mode, and then as many MS2 scans as possible were performed for the selected precursor. MS / MS analysis data were integrated and analyzed using IP2 software based on the Uniprot database. Specifically, data were recognized as peak units, information such as mass / charge (m / z) of the peaks were extracted, and then information such as amino acid sequences were confirmed from the database, and protein identification was performed by comparing them with the spectra of the obtained peptides. Here, the precursor mass error was set to 4.5 ppm, the fragment ion mass error was set to 20 ppm, and the false discovery rate (FDR) was set to 0.01 for peptide spectrum matching and protein identification using the target decoy strategy.

[0058] [Experimental Results]

[0059] 1. Development of devices for ultrasonic / high frequency processing

[0060] To confirm the effect of ultrasound / radiofrequency on advanced glycation end products (AGEs), we first designed and developed a device capable of applying ultrasound / radiofrequency to an in vitro model system. The device is a prototype developed in-house in the laboratory that can precisely control the SATA intensity, duty cycle, etc. of sound waves. Afterwards, the function was verified by measuring and confirming the output voltage, output pressure, and SATA intensity delivered to the sample according to the input voltage / duty cycle through a PZT (piezoelectric transducer). The SATA (spatial average-temporal average) intensity according to the input voltage and duty cycle of the developed device is shown in Figure 1.

[0061] 2. Application to in vitro aging tissue models and aging mouse models

[0062] Utilizing a multilayered in vitro aging model, we aimed to investigate the effects of ultrasound / radiofrequency on advanced glycation end products (AGEs) accumulated and formed in in vitro tissues due to glycation. The intended effects were twofold: 1) inhibition of AGE formation (delaying and inhibiting glycation) and 2) removal of accumulated AGEs.

[0063] First, to confirm the effect of ultrasound / radiofrequency on the inhibition and delay of AGE formation, we controlled the SATA intensity of ultrasound delivered to an in vitro aged tissue model through the input voltage and investigated the differential effects. To this end, the control group was induced to glycate the in vitro tissue model through reaction with 500 mM ribose for one week without ultrasound treatment, and the experimental group was treated with ultrasound for 1 hour per day for a total of 7 days at a 100% duty cycle during the reaction with ribose. A schematic diagram of the experimental process is shown in Figure 2. As a result, the group with an input voltage of 31.6 Vpp showed no significant effect, but the group with 47.4 Vpp showed a significant effect. This effect was also maximal at 71.1 Vpp. This suggests that there is an effective range of ultrasound / radiofrequency intensity that is effective in inhibiting and delaying AGE formation. As a result, it was observed that the formation of advanced glycation end products was inhibited by up to about 87% compared to before ultrasound / high frequency treatment, and about 155.68 mW / cm 2 It was confirmed to have a threshold value of (47.4 Vpp). The results are shown in Fig. 3.

[0064] Next, in order to confirm the removal effect of ultrasound / high frequency on advanced glycation end products (AGEs) already accumulated in the ECM, the differential effect according to the SATA intensity of ultrasound was investigated by adjusting the input voltage in the same manner as the experimental conditions above. To this end, ultrasound with different SATA intensities was treated for 1 hour per day for a total of 7 days at a 100% duty cycle to an in vitro aged tissue model that had undergone a glycation process with 500 mM ribose for 3 weeks, and the differential effect on the removal of advanced glycation end products (AGEs) was compared and analyzed. A schematic diagram of the experimental process is shown in Fig. 4. As a result, interestingly, as in the results of 'inhibition and delay of AGE formation', the group with an input voltage of 31.6 Vpp did not show a significant effect, but a significant effect began to be shown from the group with an input voltage of 47.4 Vpp. This effect was observed at 71.1 Vpp (342.49 mW / cm 2 ) showed the same maximum effect. This also means that there is an effective range for the removal effect of ultrasound / radiofrequency on already accumulated advanced glycation end products, and in the in vitro aging tissue model, this range was found to be the same as the effective range for inhibiting the formation of advanced glycation end products. As a result, it was observed that advanced glycation end products were removed by up to about 49% compared to before ultrasound / radiofrequency treatment, and 47.4 Vpp (155.68 mW / cm 2 ) was confirmed to have a threshold value. The results are shown in Fig. 5.

[0065] To quantitatively evaluate the effect of ultrasound / radiofrequency on removing AGEs, mass spectrometry was used to compare AGEs in in vitro tissue models before and after ultrasound / radiofrequency treatment. A schematic diagram of the experimental process is shown in Figure 6, and the test results are shown in Figure 7. The mass spectrometry results showed that the amount of AGEs was significantly reduced after ultrasound / radiofrequency irradiation in all of the top 15 most glycated ECM proteins in the in vitro aged tissue model. Representatively, a decrease in CML-type AGEs binding to lysine residues was observed in type 1 collagen, and a decrease in CML-type AGEs binding to lysine residues and G-DH-type AGEs binding to arginine residues was observed in fibronectin. However, the levels did not decrease to the levels of in vitro normal tissue models without glycation, indicating that AGEs were not completely removed, which is consistent with the previous results. In particular, the amount of accumulated advanced glycation end products (AGEs) relative to total substrate protein was significantly reduced from 22.6% to 7.3%. Furthermore, the removal of these AGEs was observed to be reduced across the board, regardless of amino acid residues such as lysine and arginine or AGE type, suggesting that this removal effect acts nonspecifically on multiple AGEs. The results are shown in Figure 8.

[0066] Next, based on the results of mass spectrometry, we analyzed the amino acid residue positions where advanced glycation end products (AGEs) were removed in type 1 collagen and fibronectin, which are representative structural proteins that constitute the in vitro aging tissue model before and after ultrasound / radiofrequency treatment. Analysis including both lysine and arginine residues showed that the number of sites where advanced glycation end products (AGEs) were detected decreased by approximately 29% from 21 to 15 in type 1 collagen, and by approximately 17% from 94 to 78 in fibronectin. Looking at it in detail, in type 1 collagen, AGEs at lysine residues decreased by 10% from 10 to 9, and at arginine residues decreased by approximately 45% from 11 to 6. In fibronectin, lysine residues decreased by approximately 22% from 45 to 35, and arginine residues decreased by approximately 12% from 49 to 43. Although no significant differences in the removal of advanced glycation end products (AGEs) were observed across amino acid residues, these results suggest that residues modified by AGEs were significantly removed from the triple-helical region of type I collagen, which contains cell adhesion motifs, and the type III domain of fibronectin. This suggests that chemical damage that interferes with interactions with cell surface receptors such as integrins may have been preferentially alleviated by ultrasound / radiofrequency stimulation. The results are presented in Figures 9 and 10.

[0067] Next, using a mouse model, we irradiated dorsal skin tissue with ultrasound / radiofrequency under various conditions (sound intensity, treatment time, mouse age) once a day for a total of 5 days under 100% duty cycle conditions, and verified the reproducibility of the AGE removal effect. Furthermore, we aimed to confirm the differential effects according to ultrasound / radiofrequency conditions and, based on this, derive the optimal ultrasound treatment conditions. First, as a result of irradiating ultrasound for 1 hour daily for 5 days on 18-month-old aged mice, we observed a gradual decrease in AGEs in skin tissue as the SATA intensity increased from 155 → 862 → 1,660 mW / cm². In particular, under the 1,660 mW / cm² condition, a significant reduction of approximately 28% was observed compared to the control group, and the difference was statistically significant. In addition, while there was no significant difference between the control and sham groups, the ultrasound / radiofrequency irradiation group showed a significant difference compared to the sham group. This suggests that the removal of advanced glycation end products is induced purely by acoustic stimulation, rather than by simple external treatment or environmental factors. The results are shown in Figure 11.

[0068] Next, while keeping the SATA intensity fixed at 1,660 mW / cm² and under the same conditions as the previous settings, the daily ultrasound / radiofrequency irradiation time was varied to 10 minutes, 30 minutes, and 60 minutes to evaluate the differential effects according to the treatment time. As a result, it was confirmed that as the daily irradiation time increased from 10 minutes → 30 minutes → 60 minutes, the AGE burden in the aged skin tissue gradually decreased by approximately 22%, 27%, and 42%, respectively. In addition, there was no statistically significant difference (p = 0.1696) in the 10-minute irradiation condition, but a statistically significant decrease in AGE was observed in the 30-minute irradiation conditions (p = 0.0206) and 60-minute irradiation conditions (p = 0.0060). Through this, it was confirmed that a daily exposure time of at least 30 minutes is required to effectively remove advanced glycation end products under an acoustic intensity of 1,660 mW / cm². The results are shown in Fig. 12.

[0069] Finally, this time, we tried to confirm the differential effects of ultrasound / radiofrequency according to mouse age by targeting young (6 months old), middle-aged (12 months old), and aged (18 months old) mice under the same conditions as the previous setting with the SATA intensity fixed at 1,660 mW / cm². As a result, a significant decrease in advanced glycation end products in skin tissue was observed compared to the control group in mouse models of all ages, and in particular, the aged mouse group showed the largest decrease of approximately 33% (p = 0.0002). The young group showed a decrease of approximately 28% (p = 0.0029), and the middle-aged group showed a decrease of approximately 19% (p = 0.0070). Meanwhile, no statistically significant difference was observed in the Sham group compared to the control group in any age group, suggesting that ultrasound / radiofrequency stimulation can induce AGE removal, regardless of age or the degree of AGE accumulation, although the effect may vary. The results are shown in Figure 13.

[0070] The above results of the present invention demonstrate a clear effect of ultrasound / high frequency on delaying glycation and removing advanced glycation end products.

[0071] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0072] The method for removing advanced glycation end products within a cell of the present invention is expected to be used for the prevention, improvement, or treatment of chronic diseases related to aging, as it not only suppresses the production of advanced glycation end products but also has an excellent effect of promoting the decomposition of already produced advanced glycation end products.

Claims

1. A method for removing advanced glycation end products, comprising the step of injecting wave energy into skin tissue.

2. In paragraph 1, A method for removing advanced glycation end products, wherein the above wave energy is energy having an input voltage of 47.4 Vpp to 71.1 Vpp.

3. In paragraph 1, The above wave energy is 155.68 mW / cm 2 1,660 mW / cm 2 A method for removing advanced glycation end products, which are energy sources.

4. In paragraph 1, A method for removing advanced glycation end products, wherein the above wave energy is energy injected with a duty cycle of 100%.

5. In paragraph 1, The above advanced glycation end products are Fibronectin, HeparanSulfate Proteoglycan, EMILIN-1 (Elastin microfibril interface-located protein 1), Fibulin-2, Tenascin-C, Collagen alpha-1(XII) chain, Collagen alpha-2(VI) chain, Collagen alpha-1(I) chain, Fibrillin-1, Annexin A2, Collagen alpha-1(I) chain, Collagen alpha-1(VI) chain, Transglutaminase-2, EMILIN-2 (Elastin A method for removing advanced glycation end products, wherein the advanced glycation end products are at least one selected from the group consisting of microfibril interface-located protein 2), and collagen alpha-1(XVI) chain.

6. In paragraph 1, A method for removing advanced glycation end products, wherein the removal of the advanced glycation end products is to suppress the production of advanced glycation end products or to promote the decomposition of advanced glycation end products already produced.

7. A device for preventing or improving skin aging, comprising an injection unit that injects wave energy into skin tissue.

8. In paragraph 7, A device for preventing or improving skin aging, wherein the above wave energy is energy having an input voltage of 47.4 Vpp to 71.1 Vpp.

9. In paragraph 7, A device for preventing or improving skin aging, wherein the above wave energy is 155.68 mW / cm2 to 1,660 mW / cm2.

10. In paragraph 7, A device for preventing or improving skin aging, wherein the above wave energy is energy injected with a duty cycle of 100%.

11. In paragraph 7, A device for preventing or improving skin aging, wherein the above skin aging is skin oxidation, skin dryness, skin inflammation, skin pigmentation, loss of skin elasticity, or formation of skin wrinkles.

12. A method for preventing or improving skin aging, comprising the step of injecting wave energy into skin tissue.

13. In paragraph 12, A method for preventing or improving skin aging, wherein the above wave energy is energy having an input voltage of 47.4 Vpp to 71.1 Vpp.

14. In paragraph 12, The above wave energy is 155.68 mW / cm 2 1,660 mW / cm 2 A method for preventing or improving skin aging, which is the energy of .

15. In paragraph 12, A method for preventing or improving skin aging, wherein the above wave energy is energy injected with a duty cycle of 100%.

16. In paragraph 12, A method for preventing or improving skin aging, wherein the above skin aging is skin oxidation, skin dryness, skin inflammation, skin pigmentation, loss of skin elasticity, or formation of skin wrinkles.

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