Application of antioxidative peptide Antioxidin-RL in preparation of animal cornea related products and animal model

Abstract

The invention belongs to the field of polypeptides, and particularly relates to application of antioxidative peptide Antioxidin-RL in preparation of animal cornea related products and an animal model. The technical problem to be solved by the invention is how to realize anti-aging of animal corneas. The invention provides an application of the antioxidative peptide Antioxidin-RL in delaying animal corneal aging, inhibiting animal corneal inflammation, reducing animal corneal cell DNA damage, inhibiting animal corneal mitochondrial dysfunction, and / or inhibiting a cGAS-STING signal channel in animal corneal cells, and particularly provides an application of the antioxidative peptide Antioxidin-RL in delaying animal corneal aging, inhibiting animal corneal inflammation, reducing animal corneal cell DNA damage, inhibiting animal corneal mitochondrial dysfunction and / or inhibiting animal corneal mitochondrial dysfunction. According to the application disclosed by the invention, the application is proved by carrying out antioxidative peptide Antioxidin-RL administration on a target animal, the particularity of the antioxidative peptide Antioxidin-RL in delaying animal corneal aging is disclosed, and precious resources are provided for exploring an aging mechanism of delaying age-related ocular surface diseases by the antioxidative peptide Antioxidin-RL.

Description

Technical Field

[0001] The invention belongs to the field of polypeptides, and specifically relates to the application of an antioxidant peptide Antioxidin-RL in the preparation of animal cornea-related products and an animal model. Background Art

[0002] Corneal aging is the gradual degeneration of corneal function and structure due to a variety of factors. Currently known factors of corneal aging include: long-term exposure to ultraviolet radiation causes corneal cell damage, which in turn leads to corneal aging; certain drugs, such as opioids, carteolol hydrochloride, glucocorticoids, etc., may cause corneal damage and aging; high-sugar environment can cause corneal cell damage, which in turn leads to corneal aging; with age, the function and structure of corneal cells will gradually degenerate, leading to corneal aging.

[0003] The mechanisms of corneal aging are diverse and closely related to the causes of aging. Ultraviolet radiation, drugs, etc. can cause oxidative stress in corneal cells, leading to corneal damage and aging; corneal cell apoptosis can lead to the degradation of corneal function and structure, and ultimately lead to corneal aging; ROS damage to corneal cells leads to corneal dysfunction and then corneal aging; some long-term chronic inflammatory reactions can also lead to corneal aging; changes in aging-related genes in corneal cells can also aggravate corneal cell aging.

[0004] Drugs used to treat corneal aging mainly include corneal nourishing solution used to provide necessary nutrients and maintain corneal health, anti-inflammatory drugs used to control inflammatory responses, and antioxidants used to control oxidative stress. However, these drugs have obvious deficiencies and disadvantages. Most drugs can only temporarily improve corneal function and cannot fundamentally solve the problem of corneal aging. Some drugs may cause side effects. The high price of some drugs may bring financial burden to patients. Therefore, it is very important to develop new and more effective drugs to treat corneal aging.

[0005] The cGAS-STING signaling pathway is a signaling pathway related to the body's innate immunity. The function of the cGAS-STING signaling pathway is to monitor abnormal DNA in cells and initiate downstream life activities to remove abnormal DNA. Abnormal expression of this signaling pathway will activate inflammatory responses in cells, aggravate cell damage, and lead to cell aging. Regulating the cGAS-STING signaling pathway is a potential method for corneal anti-aging.

[0006] The amphibian-derived antioxidant peptide Antioxidin-RL is a natural active peptide found in plateau frogs. It is a short peptide composed of 14 amino acids (AMRLTYNRPCIYAT). It is closely related to the fact that plateau frogs can maintain normal survival of surface cells despite living in plateau areas with high ultraviolet intensity for a long time. The peptide has strong reducing properties and can remove ROS in cells, maintain the normal working state of mitochondria in cells, and reduce the damage and spillover of mitochondrial DNA. CN101824077A was separated and purified to obtain the green frog antioxidant peptide Antioxidin-RL. Through ABTS + The scavenging method test proves that it has a very significant free radical scavenging effect, but its specific application scenarios are not disclosed, nor is it explored whether it can be used for cell anti-aging. The polypeptide OA-GI13 disclosed in CN114644705B and the polypeptide OM-GL15 disclosed in CN111704652B are similar to Antioxidin-RL and are derived from stinky dolls. They have been shown to be able to resist acute light damage to the skin.

[0007] Currently, there is no prior art that discloses or proves polypeptides that can be used for corneal anti-aging. Summary of the invention

[0008] The technical problem to be solved by the present invention is how to achieve anti-aging of animal cornea, specifically including delaying animal corneal aging, delaying animal corneal inflammation, delaying animal corneal cell DNA damage, regulating animal corneal cell mitochondrial dysfunction, inhibiting cGAS-STING signaling pathway expression, etc.

[0009] In order to solve the above technical problems, the first aspect of the present invention provides the use of Antioxidin-RL on animal cornea or the use of preparing animal cornea-related products, wherein the use on animal cornea includes one or more of the following uses: 1) Application in delaying animal corneal aging; 2) Application in inhibiting corneal inflammation in animals; 3) Application in reducing DNA damage in animal corneal cells; 4) Application in inhibiting abnormal mitochondrial function in animal cornea; 5) Application in inhibiting the cGAS-STING signaling pathway in animal corneal cells.

[0010] The application of preparing animal cornea-related products includes one or more of the following applications: 6) Application in the preparation of products related to delaying animal corneal aging; 7) Application in the preparation of products related to inhibiting animal corneal inflammation; 8) Application in the preparation of products related to reducing DNA damage in animal corneal cells; 9) Application in the preparation of products related to inhibiting abnormal mitochondrial function of animal corneal cells; 10) Application in the preparation of products related to inhibiting cGAS-STING signaling pathway in animal corneal cells; The amino acid sequence of Antioxidin-RL is AMRLTYNRPCIYAT (SEQ ID NO: 1).

[0011] Furthermore, the related product is medicine or food.

[0012] Furthermore, the animals are mainly mammals, such as humans, rabbits or mice.

[0013] The inhibition of inflammation in animal or / and mammalian cells may specifically be regulating the expression of inflammation-related genes such as IL-6, IL-8, IL-10, CCL-27, and TNF-α. The inhibition of abnormal mitochondrial function in animals or / and animal cells may specifically be regulating the expression of mitochondrial function genes such as p16, cGAS, STING, p65, and p-p65.

[0014] Furthermore, in the above application, the amphibian-derived antioxidant peptide Antioxidin-RL is administered to the animal or / and animal cells to achieve any of the following applications on the animal cornea: 1) Application in delaying animal corneal aging; 2) Application in inhibiting corneal inflammation in animals; 3) Application in reducing DNA damage in animal corneal cells; 4) Application in inhibiting abnormal mitochondrial function in animal cornea; 5) Application in inhibiting the cGAS-STING signaling pathway in animal corneal cells.

[0015] Furthermore, in the above application, the amphibian-derived antioxidant peptide Antioxidin-RL is administered to the animal cells by culturing the animal cells in a culture medium containing the amphibian-derived antioxidant peptide Antioxidin-RL, wherein the concentration of the amphibian-derived antioxidant peptide Antioxidin-RL in the culture medium is 2 μM to 200 μM.

[0016] Furthermore, the concentration of the amphibian-derived antioxidant peptide Antioxidin-RL is 15 μM, 30 μM or 60 μM.

[0017] Furthermore, the cell may be one of a corneal endothelial cell, a corneal stromal cell and a corneal epithelial cell.

[0018] Furthermore, the senescence induction of the cells is performed with an irradiation dose of 50-200 mJ / cm 2 The corneal endothelial cells, corneal stromal cells or corneal epithelial cells can be used for screening and effect verification of anti-aging products.

[0019] Furthermore, in the above application, any of the following applications can be achieved by topically administering the amphibian-derived antioxidant peptide Antioxidin-RL to the animal: 1) Application in delaying animal corneal aging; 2) Application in inhibiting corneal inflammation in animals; 3) Application in reducing DNA damage in animal corneal cells; 4) Application in inhibiting abnormal mitochondrial function in animal cornea; 5) Application in inhibiting the cGAS-STING signaling pathway in animal corneal cells.

[0020] Furthermore, in the above method, the induction of corneal aging in rats is performed by irradiating the cornea once every two days with a dose of 50-200 mJ / cm 2 Induced by UVB irradiation.

[0021] Furthermore, in the above method, corneal aging of rats was induced by irradiation once every two days at a dose of 150 mJ / cm 2 Induced by UVB irradiation.

[0022] Furthermore, in the above method, the topical ocular dosage of the amphibian-derived antioxidant peptide Antioxidin-RL is 100-1000 mg / kg / week.

[0023] In one embodiment of the present invention, the duration of topical ocular administration is 2 months.

[0024] Furthermore, topical ocular administration of the amphibian-derived antioxidant peptide Antioxidin-RL to the animal can be achieved by treating the animal with physiological saline eye drops containing the amphibian-derived antioxidant peptide Antioxidin-RL.

[0025] The animals are mainly mammals, such as New Zealand white rabbits and mice.

[0026] In one embodiment of the present invention, the topical ocular administration method is to add the drug to physiological saline eye drops to treat the animal.

[0027] The reducing or down-regulating inflammation of animals or / and animal corneal cells specifically refers to reducing or down-regulating the expression of inflammation-related genes such as IL-6, IL-8, IL-10, CCL-27, and TNF-α.

[0028] The second aspect of the present invention also provides a method for preparing an animal model, the method comprising: topically administering an amphibian-derived antioxidant peptide Antioxidin-RL to the eyes of a target animal to obtain an animal with altered gene expression levels.

[0029] Furthermore, the target animal is an animal that has been subjected to corneal aging induced by stress factors such as ultraviolet radiation, drugs, and high-sugar environment. The animal can be a rat, such as a Sprague Dawley rat.

[0030] Furthermore, in the above method, the target animal is a rat for inducing corneal aging, and the induction of corneal aging in the rat is performed by irradiating the rat with a dose of 50-200 mJ / cm2 once every two days. 2 Induced by UVB irradiation.

[0031] Furthermore, in the above method, the induction of corneal aging in rats was performed by irradiating the cornea once every two days with a dose of 150 mJ / cm 2 Induced by UVB irradiation.

[0032] Furthermore, in the above method, the topical ocular dosage of the amphibian-derived antioxidant peptide Antioxidin-RL is 100-1000 mg / kg body weight / week.

[0033] Furthermore, in the above method, the topical ocular dosage of the amphibian-derived antioxidant peptide Antioxidin-RL is 175 μg / kg body weight / week.

[0034] Furthermore, topical ocular administration of the amphibian-derived antioxidant peptide Antioxidin-RL to the target animal can be achieved by treating the animal with physiological saline eye drops containing the amphibian-derived antioxidant peptide Antioxidin-RL.

[0035] In one embodiment of the present invention, the topical ocular administration method is to add the drug to physiological saline eye drops to treat the target animal.

[0036] The duration of the topical ocular administration may be 2 months.

[0037] The animal with altered gene expression level has one or more of the following changes compared to the target animal: A) The ocular surface muscles become tighter and the opacity of the eyeball decreases; B) The expression of aging-related genes such as p16 is downregulated; C) The expression of multiple genes related to inflammation and immunity was downregulated, such as cGAS, STING, p65, p-p65, IL-6, IL-8, IL-10, CCL-27, and TNF-α.

[0038] The animal model prepared by the above method can be used for the study of aging mechanisms or the screening of anti-aging drugs.

[0039] In the present invention, the application or method may be an application or method for non-diagnosis and treatment purposes, such as for the purpose of studying the aging mechanism or screening or verifying the effect of anti-aging products.

[0040] Compared with the prior art, the beneficial effects of the present invention include at least: 1. The present invention evaluates the effect of Antioxidin-RL on aged corneal endothelial cells in vitro, and proves that 2μM-200μM Antioxidin-RL can promote cell self-replication and renewal, reduce DNA damage, reduce aging-related p16 gene expression, inhibit the decline of mitochondrial membrane potential, reduce the secretion of inflammatory factors, and reduce the positive rate of aging-related β-galactosidase staining. The above results indicate that Antioxidin-RL can alleviate the aging of human corneal endothelial cells.

[0041] 2. The present invention proves that Antioxidin-RL can reduce the ROS content in cells, reduce the damage of ROS to mitochondrial DNA, maintain the normal function of mitochondria, reduce the spillover of mitochondrial DNA into the cytoplasm, and then inhibit the expression of cGAS-STING signaling pathway, ultimately achieving the inhibitory effect on corneal cell aging.

[0042] 3. The present invention treated rats with aging corneal cells and found that long-term local ocular administration of Antioxidin-RL can delay the relaxation of eye muscles in aging rats and reduce the degree of turbidity in the eyeballs of aging rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is an analysis of the effect of amphibian-derived antioxidant peptide Antioxidin-RL in delaying UVB radiation-induced senescence of human corneal endothelial cells; Figure 1 A in the middle is the result of SA-β-Gal staining (scale bar = 100 μm); Figure 1 Middle B is the statistical data of SA-β-Gal positivity (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 1 Middle C is the result of immunofluorescence staining of γH2AX in human corneal endothelial cells (scale bar = 50 μm); Figure 1Middle D is a fluorescence intensity analysis of γH2AX immunofluorescence staining in human corneal endothelial cells (scale bar = 10 μm; mean ± SD, 3 biological replicates; unpaired Student's t test).

[0044] Figure 2 This is an analysis diagram of the effect of amphibian-derived antioxidant peptide Antioxidin-RL in alleviating UVB radiation-induced Ki67 expression in human corneal endothelial cells and reducing the secretion of inflammatory cytokines; Figure 2 Middle A is a graph showing the results of Ki67 immunofluorescence staining of human corneal endothelial cells (scale bar = 10 μm; mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 2 Middle B is the fluorescence intensity analysis of Ki67 immunofluorescence staining of human corneal endothelial cells (scale bar = 50 μm); Figure 2 Middle C is an analysis of IL-6 content in human corneal endothelial cells (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 2 Middle D is the analysis of IL-8 content in human corneal endothelial cells (mean ± SD, 3 biological replicates; unpaired Student's t test).

[0045] Figure 3 This is an analysis diagram of the amphibian-derived antioxidant peptide Antioxidin-RL reducing the expression of aging-related proteins SA-β-Gal and p16 by regulating the cGAS-STING signaling axis; Figure 3 A in the middle is the SA-β-Gal staining results of human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (scale bar = 100 μm); Figure 3 Middle B is the statistical analysis of the SA-β-Gal positive staining results of human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 3 Middle C is the Western-blots images of p16 protein in human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group; Figure 3D in the middle is the grayscale analysis image of p16 protein Western-blots of human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (mean ± SD, 3 biological replicates; unpaired Student's t test).

[0046] Figure 4 This is an analysis diagram of the amphibian-derived antioxidant peptide Antioxidin-RL regulating the expression of key proteins in the cGAS-STING signaling axis; Figure 4 A in the middle is the Western-blots images of cGAS and STING proteins in human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group; Figure 4 Middle B is the Western-blots images of p65 and p-p65 proteins in human corneal endothelial cells of the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group; Figure 4 Middle C is the grayscale analysis of Western-blots images of p65 and p-p65 proteins in human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 4 Figure 4D and Figure 4E are grayscale analysis images of cGAS and STING proteins in Western-blots of human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group, respectively (mean ± SD, 3 biological replicates; unpaired Student's t-test).

[0047] Figure 5 This is an analysis diagram showing that the amphibian-derived antioxidant peptide Antioxidin-RL affects the expression of aging and SASP-related genes by regulating the cGAS-STING signaling axis. Figure 5Middle A is the qRT-PCR expression analysis of p16 gene in human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 5 Middle B is the qRT-PCR expression analysis of cGAS gene in human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 5 Middle C is the qRT-PCR expression analysis of STING gene in human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 5 Middle D is the qRT-PCR expression analysis of IL-6 gene in human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 5 Middle E is the qRT-PCR expression analysis of IL-8 gene in human corneal endothelial cells in the control group, Ru.521 inhibitor group, aging group, aging group + Ru.521 inhibitor group, Antioxidin-RL group, and Antioxidin-RL + Ru.521 inhibitor group (mean ± SD, 3 biological replicates; unpaired Student's t test).

[0048] Figure 6 Analysis of the effects of amphibian-derived antioxidant peptide Antioxidin-RL on reducing UVB radiation-induced ROS content in human corneal endothelial cells and improving mitochondrial membrane potential. Figure 6 Middle A is the statistical graph of ROS content in human corneal endothelial cells in the control group, aging group, Antioxidin-RL group, and vitamin C group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 6 Middle B is the ROS fluorescent probe staining image in human corneal endothelial cells of the control group, aging group, Antioxidin-RL group, and vitamin C group (scale bar = 100 μm); Figure 6Middle C is the statistical graph of JC-1 mitochondrial membrane potential of human corneal endothelial cells in the control group, aging group, Antioxidin-RL group, and vitamin C group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 6 D in the middle is the JC-1 mitochondrial membrane potential fluorescence staining image of human corneal endothelial cells in the control group, aging group, Antioxidin-RL group, and vitamin C group (scale bar = 100 μm).

[0049] Figure 7 This is an analysis diagram of the effect of amphibian-derived antioxidant peptide Antioxidin-RL on improving UVB radiation-induced mitochondrial membrane function in human corneal endothelial cells; Figure 7 A in the middle is the staining image of mitochondrial fluorescent probe Mito tracker red of human corneal endothelial cells in the control group, aging group, Antioxidin-RL group, and vitamin C group (scale bar = 10 μm); Figure 7 Middle B is the qRT-PCR statistical graph of cytoplasmic mitochondrial DNA, mtND1, mtCO1, and D-loop genes in human corneal endothelial cells in the control group, aging group, Antioxidin-RL group, and vitamin C group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 7 Middle C is the qRT-PCR statistical graph of mitochondrial DNA copy number, mtND1, mtCO1, and D-loop genes in human corneal endothelial cells in the control group, aging group, Antioxidin-RL group, and vitamin C group (mean ± SD, 3 biological replicates; unpaired Student's t test).

[0050] Figure 8 The amphibian-derived antioxidant peptide Antioxidin-RL delays UVB radiation-induced corneal aging in rats; Figure 8 A in the middle is the photograph of the eyes of rats in the control group, aging group, Antioxidin-RL group, and Vitamin C group; Figure 8 Middle B is the Western-blotting analysis of p16 protein in corneal tissue of rats in the control group, aging group, Antioxidin-RL group, and Vitamin C group; Figure 8 Middle C is the grayscale analysis result of Western-blotting of p16 protein in corneal tissue of rats in the control group, aging group, Antioxidin-RL group, and Vitamin C group (mean ± SD, 3 biological replicates; unpaired Student's t test); Figure 8 D is the qRT-PCR analysis of p16 gene in corneal tissues of rats in the control group, aging group, Antioxidin-RL group, and vitamin C group (mean ± SD, 3 biological replicates; unpaired Student's t test). DETAILED DESCRIPTION

[0051] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0052] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0053] Example 1: Antioxidant peptide Antioxidin-RL from amphibian delays UVB radiation-induced senescence of human corneal endothelial cells The anti-aging effect of amphibian-derived antioxidant peptide Antioxidin-RL was detected in aged human corneal endothelial cells. Different concentrations of amphibian-derived antioxidant peptide Antioxidin-RL (2 μM-200 μM) were used to treat UVB radiation-induced aged human corneal endothelial cells for 4 consecutive generations. Aged human corneal endothelial cells were cultured in sterile cell culture bottles; the complete culture medium for human corneal endothelial cells consisted of DMEM basal medium (Hyclone), 10% fetal bovine serum (Gibco, Cat#10099-141), and 1% penicillin / streptomycin (Solarbio); no mycoplasma contamination was detected during the culture of human corneal endothelial cells. Amphibian-derived antioxidant peptide Antioxidin-RL synthesized by Shanghai Jier Biochemical Company was used in the cell experiment, and the solvent was DMEM basal medium.

[0054] The specific method is as follows: 1. SA-β-Gal staining Cell senescence-associated β-galactosidase staining is a method for staining and detecting senescent cells or tissues based on the upregulation of SA-β-Gal (senescence-associated beta-galactosidase) activity levels during senescence. When X-Gal is used as a substrate, cells will produce dark blue products under the catalysis of senescence-specific β-galactosidase. SA-β-Gal staining was performed on human corneal endothelial cells treated with normal control group, UVB radiation-induced senescence group and Antioxidin-RL group, respectively, and the steps are as follows: 1) Inoculate healthy human corneal endothelial cells at an appropriate density in a six-well cell plate; 2) When the cell density reaches more than 80%, discard the original culture medium and wash twice with PBS; 3) Fix with 4% paraformaldehyde for 30 minutes; 4) Wash twice with PBS; 5) SA-β-Gal staining kit (Solarbio, Cat. No. G1580), incubate overnight at 37°C in the dark; 6) Wash twice with PBS; 7) Observe and take pictures under a microscope, and further perform quantitative statistical analysis on the ratio of SA-β-Gal positive cells in the cells.

[0055] The results are as follows Figure 1 As shown in A and B, the staining results showed that the amphibian-derived antioxidant peptide Antioxidin-RL in the range of 2 μM-200 μM can effectively reduce the activity of SA-β-Gal in aged human corneal endothelial cells.

[0056] 2. Immunofluorescence staining analysis 1) Seed cells at an appropriate density in a six-well plate; 2) When the cell density reaches more than 80%, discard the original culture medium and wash twice with PBS; 3) Fix with 4% paraformaldehyde for 30 minutes; 4) Wash twice with PBS; 5) Incubate with 0.25% Triton X-100 at room temperature for 15 minutes. 6) Then switch to 3% bovine serum for blocking at room temperature for 1 hour; 7) Change to blocking solution with primary antibody and incubate overnight at 4°C (Ki67, Bioss product number: bs-2130R; γH2AX, Affinit, product number: AF8482).

[0057] 8) Rinse with PBS three times; 9) Then add the corresponding secondary antibody and incubate at room temperature for 1 hour (goat anti-rabbit, Bioss, catalog number: bs-0295G-AF488); 10) After rinsing three times with PBS; 11) Add nuclear staining reagent DAPI (working concentration is 2 g / ml); 12) Seal the slides and observe and photograph them under a fluorescence microscope. Further quantitative statistical analysis was performed on the ratio of Ki67 and γH2AX positive cells in the cells.

[0058] The results are as follows Figure 1 C, D and Figure 2As shown in A and B in the figure, it can be seen that the amphibian-derived antioxidant peptide Antioxidin-RL in the range of 2 μM-200 μM can effectively reduce the amount of γH2AX in aging human corneal endothelial cells and increase the activity of Ki67.

[0059] 3. ELISA The expression levels of inflammatory cytokines in the culture medium of UVB-irradiated aged human corneal endothelial cells treated with amphibian-derived antioxidant peptide Antioxidin-RL were detected by enzyme-linked immunosorbent assay. Human IL-6 and IL-8 enzyme-linked immunosorbent assay kits were purchased from ELISA, and the test results were quantitatively analyzed using an MK3 microplate reader (Thermo).

[0060] The results are as follows Figure 2 As shown in C and D, it can be seen that the amphibian-derived antioxidant peptide Antioxidin-RL in the range of 2 μM-200 μM can effectively reduce the content of inflammatory cytokines IL-6 and IL-8 in aged human corneal endothelial cells.

[0061] In general, the results of the present application indicate that the amphibian-derived antioxidant peptide Antioxidin-RL can delay the aging of human corneal endothelial cells.

[0062] Example 2: Amphibian-derived antioxidant peptide Antioxidin-RL regulates the cGAS-STING signaling axis to delay the aging of human corneal endothelial cells In order to explore the molecular mechanism of the effect of amphibian-derived antioxidant peptide Antioxidin-RL on body aging and its relationship with the cGAS-STING signaling axis, this application used cGAS inhibitor Ru.521 (10μM) to treat cells. The experimental method is as follows: 1. Perform SA-β-Gal staining in the same manner as in Example 1 The results are as follows Figure 3 As shown in A and B, it can be seen that the use of cGAS inhibitor Ru.521 and amphibian-derived antioxidant peptide Antioxidin-RL can effectively reduce the activity of SA-β-Gal in aged human corneal endothelial cells.

[0063] 2. Western blot detection of protein expression Cells were seeded at an appropriate density in a six-well plate; when the cell density reached more than 80%, the original culture medium was discarded and washed twice with PBS; cells were collected, proteins were extracted, and Western blot was performed. The primary antibodies used were p16, cGAS, STING, p65, and p-p65 antibodies (Proteintech, Catalog No.: 10883-1-AP, 26426-1-AP, 19851-1-AP, 80979-1-RR, 82335-1-RR), and the secondary antibody was goat anti-rabbit antibody (Zenbio, Catalog No.: 511203). GAPDH was used as an internal reference, the primary antibody was mouse anti-GAPDH antibody (Huabio, Catalog No.: EM1901-57), and the secondary antibody was goat anti-mouse antibody (Proteintech, Catalog No.: RGAM001).

[0064] The results are as follows Figure 3 C, D and Figure 4 As shown, it can be seen that the use of cGAS inhibitor Ru.521 and the amphibian-derived antioxidant peptide Antioxidin-RL can effectively reduce the expression levels of p16, cGAS, STING, p65, and p-p65 in aged human corneal endothelial cells.

[0065] 3. Real-time fluorescence quantitative PCR Cells were seeded at an appropriate density in a six-well plate. When the cell density reached more than 80%, the original culture medium was discarded and washed twice with PBS. The cells were collected, RNA was extracted, and real-time fluorescence quantitative PCR (Hieff qPCR SYBR Green MasterMix (Low Rox Plus), Yeasen, Cat. No.: 11202ES08) was performed. The detection primer used β-actin as the internal reference.

[0066] The results are as follows Figure 5 As shown, it can be seen that the use of cGAS inhibitor Ru.521 and Antioxidin-RL can effectively reduce the transcription levels of p16, cGAS, STING, IL-6, and IL-8 in aged human corneal endothelial cells.

[0067] Example 3: Amphibian-derived antioxidant peptide Antioxidin-RL improves UVB radiation-induced mitochondrial function of aged human corneal endothelial cells and reduces mitochondrial DNA spillover 1. Intracellular ROS detection As oxidative phosphorylation reactions occur in cells, ROS are generated during electron transfer. Large amounts of ROS will destroy the normal physiological functions of cells. During cell aging, ROS will accumulate in large quantities. The ROS content of human corneal endothelial cells was detected using a commercial cell ROS kit: Reactive Oxygen Species Kit (Solarbio, Cat. No. CA1410). The specific experimental steps are as follows: 1) Digest the test cells with trypsin and collect them in a 1.5 mL EP tube.

[0068] 2) Prepare the ROS working solution from the stock solution in the ROS detection kit.

[0069] 3) Add ROS working solution to resuspend the cells, protect from light, and place in a cell culture incubator at 37°C and 5% CO2 for staining for 30 minutes.

[0070] 4) Detect ROS in human corneal endothelial cells using flow cytometry (BD LSRFortessa). The intensity of green fluorescence in the cells indicates the intracellular ROS content.

[0071] The results are as follows Figure 6 As shown in A and B, it can be seen that the use of amphibian-derived antioxidant peptide Antioxidin-RL can effectively reduce the content of ROS in aging human corneal endothelial cells.

[0072] 2. Mitochondrial membrane potential detection During oxidative respiration, mitochondria store the energy generated in the form of electrochemical potential energy in the mitochondrial inner membrane. The transmembrane potential generated by the asymmetric distribution of hydrogen ions and other ions on both sides of the mitochondrial inner membrane is called the mitochondrial membrane potential. During cell aging, the membrane potential decreases. The mitochondrial membrane potential of human corneal endothelial cells was detected using the commercial JC-1 kit: Mitochondrial Membrane Potential Detection Kit (JC-1) (Solarbio, Cat. No.: M8650). The specific experimental steps are as follows: 1) Digest the test cells with trypsin and collect them in a 1.5 mL EP tube.

[0073] 2) Prepare the JC-1 working solution from the stock solution in the JC-1 test kit.

[0074] 3) Add JC-1 working solution to resuspend the cells, protect from light, and place in a cell culture incubator at 37°C and 5% CO2 for staining for 30 minutes.

[0075] 4) Flow cytometry (BD LSRFortessa) was used to detect the mitochondrial membrane potential of human corneal endothelial cells. The intensity of green and red fluorescence in the cells indicated the mitochondrial membrane potential.

[0076] The results are as follows Figure 6 As shown in C and D, it can be seen that the use of amphibian-derived antioxidant peptide Antioxidin-RL can effectively alleviate the decline in mitochondrial potential in aging human corneal endothelial cells.

[0077] 3. Real-time fluorescence quantitative PCR Cells were seeded at an appropriate density in a six-well plate. When the cell density reached more than 80%, the original culture medium was discarded and the cells were washed twice with PBS. The cells were collected, and total DNA and cytoplasmic DNA were extracted for real-time fluorescence quantitative PCR (Hieff qPCRSYBR Green Master Mix (Low Rox Plus), Yeasen, Cat. No.: 11202ES08). The detection primer used 18sDNA as the internal reference.

[0078] The results are as follows Figure 7 As shown in B and C, it can be seen that the use of amphibian-derived antioxidant peptide Antioxidin-RL can effectively reduce the overflow of mitochondrial DNA in aging human corneal endothelial cells and alleviate the reduction of mitochondrial DNA copy number.

[0079] 4. Perform immunofluorescence staining in the same manner as in Example 1 The results are as follows Figure 7 As shown in A, it can be seen that the use of amphibian-derived antioxidant peptide Antioxidin-RL can effectively increase the co-localization of dsDNA and mitochondria in aged human corneal endothelial cells and alleviate the overflow of mitochondrial DNA.

[0080] Example 4: Amphibian-derived antioxidant peptide Antioxidin-RL delays UVB radiation-induced corneal endothelial aging in rats and improves oxidative stress In order to test the anti-aging effect of amphibian-derived antioxidant peptide Antioxidin-RL in vivo, the irradiation dose of 150 mJ / cm was used every two days. 2 Rat eyes were irradiated with UVB to induce corneal aging. After each irradiation, 175 μg / kg body weight / week of amphibian-derived antioxidant peptide Antioxidin-RL was added to alleviate the symptoms of corneal aging in rats. Various aging indicators were detected after two months of continuous treatment.

[0081] 1. Observation of rat ocular surface The differences in the eyes of rats after aging treatment and rats treated with amphibian-derived antioxidant peptide Antioxidin-RL were observed and photographed to record the degree of turbidity.

[0082] The results are as follows Figure 8As shown in A, it can be seen that the use of amphibian-derived antioxidant peptide Antioxidin-RL can effectively alleviate the opacity of the eye and relaxation of the ocular surface muscles caused by aging of the rat eye tissue.

[0083] 2. Detect protein expression by Western blot.

[0084] Rat corneal tissue was obtained, washed and protein was extracted. Western blot was performed. The primary antibody used was p16 antibody (Proteintech, Catalog No.: 10883-1-AP), and the secondary antibody was goat anti-rabbit antibody (Zenbio, Catalog No.: 511203). GAPDH was used as an internal reference, the primary antibody was mouse anti-GAPDH antibody (Huabio, Catalog No.: EM1901-57), and the secondary antibody was goat anti-mouse antibody (Proteintech, Catalog No.: RGAM001).

[0085] The results are as follows Figure 8 As shown in B and C, it can be seen that the use of amphibian-derived antioxidant peptide Antioxidin-RL can effectively reduce the expression of aging-related protein p16 in rat eye tissue 3. Real-time fluorescence quantitative PCR Rat corneal tissue was obtained, washed and RNA was extracted, and real-time fluorescence quantitative PCR (Hieff qPCR SYBR Green Master Mix (Low Rox Plus) was performed. The detection primer used β-actin as the internal reference.

[0086] The results are as follows Figure 8 As shown in D, it can be seen that the use of amphibian-derived antioxidant peptide Antioxidin-RL can effectively reduce the transcription of the aging-related gene p16 in rat eye tissue.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. Application of the antioxidant peptide Antioxidin-RL in the preparation of animal cornea-related products, characterized in that: The application of the preparation of animal cornea-related products includes one or more of the following applications: application in the preparation of products related to delaying animal corneal aging, application in the preparation of products related to inhibiting animal corneal inflammation, application in the preparation of products related to reducing animal corneal cell DNA damage, application in the preparation of products related to inhibiting animal corneal cell mitochondrial dysfunction, and application in the preparation of products related to inhibiting the cGAS-STING signaling pathway in animal corneal cells.

2. The use according to claim 1, characterized in that: The related products are medicines or foods.

3. The use according to claim 1, characterized in that: The animals are humans, rabbits and mice.

4. The use according to claim 1, characterized in that: The animal corneal cell is one of a corneal endothelial cell, a corneal stromal cell and a corneal epithelial cell.

5. A method for preparing an animal model, characterized in that: The method comprises: topically administering the antioxidant peptide Antioxidin-RL to the eyes of the target animal to obtain the animal with the changed gene expression level.

6. The method for preparing an animal model according to claim 5, characterized in that: The target animal is an animal whose corneal aging is induced by ultraviolet radiation, drugs or high sugar environment.

7. The method for preparing an animal model according to claim 6, characterized in that: The target animal is a rat that is induced to have corneal aging, and the irradiation dose is 150 mJ / cm2 once every two days. 2 Induced by UVB irradiation.

8. The method for preparing an animal model according to claim 7, characterized in that: The topical ocular dosage of the antioxidant peptide Antioxidin-RL is 100-1000 mg / kg body weight / week.

9. The method for preparing an animal model according to claim 8, characterized in that: The animal with altered gene expression level has one or more of the following changes compared to the target animal: A) The ocular surface muscles become tighter and the opacity of the eyeball decreases; B) The expression of aging-related genes such as p16 is downregulated; C) The expression of multiple genes related to inflammation and immunity was downregulated, such as cGAS, STING, p65, p-p65, IL-6, IL-8, IL-10, CCL-27, and TNF-α.

10. An animal model, characterized in that: Prepared according to the method for preparing an animal model according to any one of claims 5 to 9.

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