Method for evaluating senescence of dermal fibroblasts

Abstract

The invention discloses a method for evaluating senescence of dermal fibroblasts. The method comprises the following steps: measuring the assembly density of F-actin in dermal fibroblasts, and comparing with a contrast; if the assembly density is lower than that of the contrast, evaluating that the dermal fibroblasts are senescent cells or senescent cells; the control includes the assembly density of F-actin in dermal fibroblasts that are defined to be unaged. The evaluation method has the effect of screening out symptoms such as skin wrinkles, relaxation and droop caused by the change of the physical and chemical environment of the extracellular matrix.

Description

Technical Field

[0001] This application relates to the field of skin repair technology, specifically to a method for evaluating dermal fibroblast senescence. Background Technology

[0002] The mechanical interaction between cells and the extracellular matrix (ECM) is crucial for tissue homeostasis, while aging ECM and cells can interact to promote the aging process. Changes in the extracellular matrix are one of the 14 major indicators of aging (Cell 2025, 188(8): 2043-2062). In young dermal ECM, intact collagen fiber bundles are densely arranged, and fibroblasts physically attach to intact collagen fibers through specific cell surface integrins (…). Figure 12 (A) This allows dermal fibroblasts to exert tension on surrounding collagen fibers through the mechanical forces generated by cytoskeleton assembly, creating prestress within the ECM network and establishing appropriate skin tension. During skin aging, dermal ECM fragmentation and decreased matrix viscoelasticity restrict fibroblast attachment within the matrix. Figure 12 (B) Fibroblasts (in the middle layer of the skin) influence the generation of cellular mechanomechanical forces, thereby altering cell morphology and function, and potentially accelerating cellular senescence. Because the mechanical properties of tissues are closely coupled and interdependent with the physiological functions embedded in fibroblasts, understanding skin aging requires understanding the biophysical characteristics (mechanical properties) of aging dermal fibroblasts. However, existing reports have not clearly defined the regulatory relationship between fibroblast mechanomechanical forces and cellular senescence.

[0003] Existing methods for measuring cellular senescence, such as SA-β-Gal staining, p16 / p21 protein detection, and DNA damage focal point (γ-H2AX) detection, all detect the "endpoint" state after senescence has occurred. However, they cannot reveal how cellular mechanical properties (such as contractility, stiffness, and the ability to sense and remodel the matrix) gradually fail during the senescence process. Since senescence is a dynamic process, the aforementioned methods cannot provide early warning of this early and continuous decline in biophysical function, thus missing the golden window for early intervention.

[0004] Therefore, there is an urgent need in this field for a novel method to evaluate cellular senescence. Summary of the Invention

[0005] Based on this, this application provides at least one method for evaluating dermal fibroblast senescence.

[0006] In a first aspect of this application, a method for evaluating the senescence state of dermal fibroblasts is provided, the method comprising:

[0007] The assembly density of F-actin in dermal fibroblasts was measured and compared with that of a control.

[0008] If the assembly density is lower than that of the control, the dermal fibroblasts are evaluated as senescent cells or senescent cells.

[0009] The control group included the assembly density of F-actin in dermal fibroblasts that were definitively identified as non-senescent.

[0010] In a second aspect of this application, a method is provided for evaluating the effect of an analyte on inhibiting and / or improving dermal fibroblast senescence, the method comprising:

[0011] The test substance was mixed with senescent dermal fibroblasts and incubated.

[0012] The assembly density of F-actin and changes in cell morphology in dermal fibroblasts were measured before and after treatment.

[0013] In a third aspect of this application, the use of a substance that maintains F-actin cytoskeleton homeostasis in the preparation of an agent that inhibits and / or improves cellular senescence is provided.

[0014] In a fourth aspect of this application, a method is provided for screening agents that inhibit and / or improve dermal fibroblast senescence, the method comprising:

[0015] The candidate material was mixed with senescent dermal fibroblasts and incubated.

[0016] The assembly density of F-actin and changes in cell morphology in dermal fibroblasts were measured before and after treatment.

[0017] One aspect of this application demonstrates that cytoskeleton mechanics can directly influence aging phenotypes by inversely modulating the mechanical properties of senescent cells. Intrinsic cellular forces (F-actin cytoskeleton parameters) can serve as a novel quantitative indicator of cellular senescence. This method breaks through traditional endocrinology research and fills the gap in the interdisciplinary field of "cellular mechanics-aging." Compared to other aging indicators that are singularly directional at the cellular level, the evaluation method in this application possesses both subcellular microscopic and "macroscopic" correlations. F-actin cytoskeleton parameters reflect the cell's own mechanics (such as Young's modulus) and its ability to interact with the surrounding matrix. This evaluation method has the potential to screen for effective countermeasures against symptoms such as wrinkles, sagging, and drooping caused by changes in the physicochemical environment of the extracellular matrix. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0019] Figure 1 This application illustrates a method for characterizing senescent fibroblasts under UVB radiation using β-galactosidase staining (One-way ANOVA test). p<0.01, A schematic diagram of the results (p<0.001).

[0020] Figure 2 This application shows a method for characterizing fibroblast aging under UVB radiation using the P21 (One-way ANOVA test) indicator in one embodiment of the present application. p<0.01).

[0021] Figure 3 This application shows the morphological changes of senescent fibroblasts due to UVB radiation in one embodiment of the present application (One-way ANOVA test). p<0.001).

[0022] Figure 4 This invention illustrates the change in Young's modulus of senescent fibroblasts under UVB radiation (using a one-way ANOVA test) in one embodiment of this application. p<0.001).

[0023] Figure 5 This invention demonstrates the changes in the ability of senescent fibroblasts to shrink the matrix under UVB radiation in one embodiment (One-way ANOVA test). p<0.01, p<0.001).

[0024] Figure 6 This application demonstrates changes in motor proteins (MLC, p-MLC) within senescent fibroblasts due to UVB radiation in one embodiment (using a one-way ANOVA test). p<0.05, p<0.01).

[0025] Figure 7This invention illustrates changes in cell morphology and Young's modulus (One-way ANOVA test) after impaired F-actin cytoskeleton assembly in primary dermal fibroblasts (HDF) according to one embodiment of this application. p<0.01, p<0.001).

[0026] Figure 8 This invention illustrates the change in the senescence phenotype marker P21 in primary dermal fibroblasts (HDF) after impaired F-actin cytoskeleton assembly in one embodiment of this application (using a one-way ANOVA test). p<0.05, p<0.01).

[0027] Figure 9 This invention illustrates cellular morphological changes and changes in the aging marker P21 in radiation-protected senescent fibroblasts during F-actin cytoskeleton assembly, as well as changes in these changes (using a one-way ANOVA test) according to one embodiment of the present application. p<0.01).

[0028] Figure 10 This application demonstrates the protective effect of cyclic peptide 163 on the morphology of radiation-aged fibroblasts and the protection of F-actin assembly in one embodiment.

[0029] Figure 11 This application demonstrates the effect of cyclic peptide 163 on slowing down the aging of radiation-senescent fibroblasts in one embodiment (One-way ANOVA test). p<0.001).

[0030] Figure 12 This invention illustrates the mechanical interaction (AB) between dermal fibroblasts and ECM in young and old skin, and the generation of mechanical forces in young dermal fibroblasts (C) in one embodiment of this application. Detailed Implementation

[0031] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0033] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0034] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0035] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0036] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0037] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0038] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0039] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0040] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0041] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0042] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0043] One of the objectives of this application is to provide a novel method for evaluating fibroblast aging and functional regulators based on a new interdisciplinary paradigm of "mechanics-biology," leveraging the regulatory relationship between the micromechanical properties of fibroblasts and extrinsic aging. This aims to offer a new scientific perspective and intervention strategy for skin anti-aging.

[0044] To achieve the above objectives, a systematic analysis of changes in the mechanical properties of photosenescent fibroblasts revealed a bidirectional regulatory relationship between fibroblast mechanical properties and aging. The inventors have innovatively revealed that intracellular mechanical disturbances / damage are a triggering factor for the appearance or exacerbation of aging phenotypes. Figure 12Based on these findings, the inventors have developed the mechanical properties of dermal fibroblasts (F-actin cytoskeleton parameters) as a novel quantitative indicator for assessing skin aging and as a screening method for cellular aging / functional regulators.

[0045] The inventors first tested the changes in the mechanical characteristics of UV-aged fibroblasts and analyzed the changes in the mechanical factors (the contents of the actomyosin skeleton tension). They found that UV-aged fibroblasts had a decreased Young's modulus, flattened cell morphology, an imbalance in the actomyosin skeleton tension, and significantly impaired ability to contract the matrix.

[0046] Next, the key role of the mechanotropic factor F-actin in fibroblast senescence was evaluated.

[0047] Finally, the test substance was applied to photoaged fibroblasts, and the effect of the test substance on delaying cell aging was evaluated based on the assembly density of the scaffold content F-actin and cell morphology.

[0048] In the above process, photoaging of dermal fibroblasts is induced by single or multiple UVB radiation. Related aging markers such as β-galactosidase and cell cycle inhibitor P21 increase.

[0049] The morphological changes of dermal fibroblasts due to photoaging are characterized by a change in cell shape from elongated spindle-shaped to polygonal, and cell flattening.

[0050] The characteristics of photoaging changes in the mechanical forces of dermal fibroblasts are: impaired tension of the actomyosin cytoskeleton, reduced Young's modulus of cells, and significant loss of the ability of cells to contract collagen fiber matrix.

[0051] Among the contents of the actosomal skeleton tension (key factors of cellular mechanodynamics: F-actin and myosin): F-actin is expanded, its density is reduced, and its distribution is uneven; myosin expression is reduced and its activity is decreased.

[0052] In radiation-induced aging dermal fibroblasts, impaired F-actin assembly reduces Young's modulus, accelerates the appearance of aging phenotypes, and increases the expression of cell cycle repressor protein P21.

[0053] Maintaining F-actin assembly effectively through drugs can improve cellular senescence phenotypes, manifested as: protection of long spindle-shaped cell morphology and reduced expression of P21, a marker of senescence.

[0054] Furthermore, adding any test substance that effectively maintains F-actin cytoskeleton homeostasis to radiation-induced senescent cells can effectively protect cell morphology and improve cellular senescent phenotypes.

[0055] I do not wish to be limited by any theory, but I believe that "F-actin cytoskeleton homeostasis" refers to the assembly and disassembly, organization and mechanical properties of F-actin, which can be coordinated with the needs inside and outside the cell. For example, it can be manifested as a balance between polymerization and depolymerization, that is, the continuous and controlled conversion between monomeric G-actin and fibrillary F-actin.

[0056] One aspect of this application provides a method for evaluating the senescence state of dermal fibroblasts, the method comprising:

[0057] The assembly density of F-actin in dermal fibroblasts was measured and compared with that of a control.

[0058] If the assembly density is lower than that of the control, the dermal fibroblasts are evaluated as senescent cells or aged cells.

[0059] The term "F-actin density" can be defined as the F-actin fluorescence intensity per unit cell area: that is, the average fluorescence intensity (Mean) = the sum of fluorescence intensities within a single cell region (IntDen) / the area of ​​that cell region (Area).

[0060] In some embodiments, the control includes the assembly density of F-actin in dermal fibroblasts that are explicitly non-senescent (e.g., in a young state).

[0061] In some embodiments, the unsenescent dermal fibroblasts include irradiated young primary dermal fibroblasts.

[0062] It should be understood that the “young state” does not mean the absence of any signs of aging, but rather a relatively unaged state characterized by combining various methods used in the art to determine cellular senescence (including but not limited to cell cycle inhibitor P21, Young’s modulus, etc.).

[0063] In some implementations, the method for evaluating the senescence status of dermal fibroblasts may also be combined with other methods for evaluating cellular senescence status, including but not limited to one or more of the following assays:

[0064] 1) Determine the cell cycle inhibitory protein P21 in dermal fibroblasts;

[0065] 2) Determine the Young's modulus of dermal fibroblasts;

[0066] 3) β-galactosidase staining;

[0067] 4) Measure collagen contraction in dermal fibroblasts; and,

[0068] 5) Cell spindle-shaped morphology.

[0069] The expression level of cell cycle repressor protein P21 can be determined by Western blotting, in accordance with conventional methods in the art.

[0070] The determination of Young's modulus of cells can be performed in accordance with conventional methods in the art, and, exemplarily, can be based on a nanoindenter using fiber optic interferometry.

[0071] Another aspect of this application provides a method for evaluating the effect of an analyte on inhibiting and / or improving dermal fibroblast senescence, the method comprising:

[0072] The test substance was mixed with senescent dermal fibroblasts and incubated.

[0073] The assembly density of F-actin and changes in cell morphology in dermal fibroblasts were measured before and after treatment.

[0074] In some implementations, if the assembly density of F-actin is increased and the cell spindle morphology is preserved before and after treatment, the analyte is determined to have the effect of inhibiting and / or improving dermal fibroblast senescence.

[0075] Referring to the above methods for evaluating the senescence status of dermal fibroblasts, this method for evaluating the effect of the analyte on inhibiting and / or improving dermal fibroblast senescence can also be combined with other methods in the art for evaluating cell senescence status, including but not limited to one or more of the following assays:

[0076] 1) Determine the cell cycle inhibitory protein P21 in dermal fibroblasts;

[0077] 2) Determine the Young's modulus of dermal fibroblasts;

[0078] 3) β-galactosidase staining;

[0079] 4) Measure collagen contraction in dermal fibroblasts; and,

[0080] 5) Cell spindle-shaped morphology.

[0081] In some embodiments, the senescent dermal fibroblasts are radiation-senescent fibroblasts.

[0082] In some embodiments, the radiation-senescent fibroblasts are fibroblasts obtained by UVB radiation-induced senescence.

[0083] In some embodiments, the method for preparing the radiation-senescent fibroblasts includes:

[0084] Primary dermal fibroblasts with a cell passage number of 6 or less were exposed to ultraviolet radiation at a dose of 120 mJ / cm². 2 Of the UVB mentioned above.

[0085] In some embodiments, primary dermal fibroblasts with a cell passage number of 6 or less are cultured in a culture environment before exposure to UVB.

[0086] In some implementations, the incubation time is more than 24 hours, for example, 24 to 48 hours.

[0087] In some implementations, the incubation time is 24 hours.

[0088] Another aspect of this application provides the use of substances that maintain F-actin cytoskeleton homeostasis in the preparation of formulations that inhibit and / or improve cellular senescence.

[0089] Another aspect of this application provides a method for screening agents that inhibit and / or improve dermal fibroblast senescence, the method comprising:

[0090] The candidate material was mixed with senescent dermal fibroblasts and incubated.

[0091] The assembly density of F-actin and changes in cell morphology in dermal fibroblasts were measured before and after treatment.

[0092] In some implementations, if the assembly density of F-actin is increased and the cell spindle morphology is protected before and after treatment, the candidate substance is determined to have the effect of inhibiting and / or improving dermal fibroblast senescence.

[0093] Similarly, this method for screening agents that inhibit and / or improve dermal fibroblast senescence can also be combined with other methods in the art for evaluating cellular senescence status, including but not limited to one or more of the following assays:

[0094] 1) Determine the cell cycle inhibitory protein P21 in dermal fibroblasts;

[0095] 2) Determine the Young's modulus of dermal fibroblasts;

[0096] 3) β-galactosidase staining;

[0097] 4) Measure collagen contraction in dermal fibroblasts; and,

[0098] 5) Cell spindle-shaped morphology.

[0099] In some embodiments, the senescent dermal fibroblasts are radiation-senescent fibroblasts.

[0100] In some embodiments, the radiation-senescent fibroblasts are fibroblasts obtained by UVB radiation-induced senescence.

[0101] In some embodiments, the method for preparing the radiation-senescent fibroblasts includes:

[0102] Primary dermal fibroblasts with a cell passage number of 6 or less were exposed to ultraviolet radiation at a dose of 120 mJ / cm². 2 Of the UVB mentioned above.

[0103] In some embodiments, primary dermal fibroblasts with a cell passage number of 6 or less are cultured in a culture environment before exposure to UVB.

[0104] In some implementations, the incubation time is more than 24 hours, for example, 24 to 48 hours.

[0105] In some implementations, the incubation time is 24 hours.

[0106] In the above methods, those skilled in the art can adjust the amount of the analyte and / or candidate substance added according to their different types.

[0107] The following are some examples.

[0108] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0109] Example 1

[0110] 1. Test the changes in the mechanical properties of photosenescent cells and assess the expression and activity of force signaling factors.

[0111] This step primarily assesses the effects of aging-induced cellular mechanical damage by detecting changes in the mechanical properties of radiation-induced senescent fibroblasts and corresponding mechanical force-inducing factors.

[0112] 1) Cell Culture: Primary human dermal fibroblasts (HDF) were cultured statically in low-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (P / S) at 37 °C in a 5% CO2 cell culture incubator, with the medium replaced with fresh cells every two days. Once the cells reached 90% confluence, they were passaged or seeded onto plates. Cells were digested and counted after each passage or seeding. The cell density for each passage was 1.6 × 10⁶ cells / year.4 ~ 2 × 10 4 / cm 2 Cells with a passage number of 6 or less are considered young HDF.

[0113] 2) UVB radiation-induced senescence: Based on the testing requirements, HDF cells were seeded in cell culture plates of different sizes (24 / 12 / 6 wells) and cultured for 24 h. The culture medium was then replaced with a small amount of PBS (covering the cells). Subsequently, the cells were placed in a UV radiation meter and exposed to a concentration of 150 mJ / cm². 2 (10 min, 0.25 mW / cm) 2 In UVB, when multiple irradiations are required, each irradiation is 24 hours apart. After the UV irradiation ends, the target index is detected after 24 hours of incubation. The group that did not receive irradiation is set as the control (CTRL). The model group that received only one irradiation is named UV-1, and the model group that received two irradiations at 24-hour intervals is named UV-2.

[0114] 3) β-Galactosidase Staining: After cell culture in 12-well cell culture plates, the cell culture medium was discarded, the cells were washed with PBS buffer, and 1 mL of fixative was added. The plates were then fixed at room temperature for 15 min. The fixative was discarded, and the cells were washed three times with PBS buffer. The working solution for the β-galactosidase staining kit was prepared according to the kit instructions and incubated. After approximately 24 h of incubation, the 12-well plates were removed, and the staining of senescent-related β-galactosidase was observed using an inverted optical microscope. The proportion of senescent cells was also counted. The results showed that UVB radiation significantly increased β-galactosidase expression in fibroblasts, and the expression level was dose-dependent on UVB radiation. (Reference: [Insert reference here]) Figure 1 .

[0115] 4) Detection of cell cycle inhibitor P21: The expression level of cell cycle inhibitor P21 was determined by Western blotting. Protease and phosphatase inhibitors were added to RIPA lysis buffer to extract total protein from control and model fibroblasts. Protein concentration was then determined using the BCA method. An appropriate amount of 5× protein loading buffer was added to each protein sample to standardize the final total protein concentration. The protein was denatured by heating in a 95°C metal bath for 5 min. Protein P21 was isolated using a 12% SDS-PAGE gel based on its molecular weight. The isolated protein was transferred to a PVDF membrane, which was then blocked with 5% skim milk at room temperature for 2–4 hours. The PVDF membrane was gently washed several times with TBST buffer to remove residual blocking solution. The corresponding primary antibody (rabbit monoclonal antibody CDKN1A / P21) was diluted with TBST and incubated overnight at 4°C. The next day, the primary antibody was aspirated, and the membrane was washed three times with TBST buffer for 10 minutes each time. Then, the corresponding species-binding HRP secondary antibody was added, and the membrane was incubated on a shaker at room temperature for 1–2 hours. The membrane was then washed three more times with TBST buffer for 10 minutes each time. Finally, the protein bands were exposed using ECL chemiluminescence working solution under a gel imaging system. The results showed that UVB radiation significantly increased the cell cycle repressor protein P21 in fibroblasts. Figure 2 . Figure 1 and Figure 2 All of these studies demonstrate that UVB radiation causes senescent fibroblasts.

[0116] 5) Cell Morphology (F-actin Assembly and Distribution): HDF was sparsely seeded into 24-well slides to facilitate the capture and identification of individual cell images. After cell culture, the cytoskeleton (F-actin) and cell nuclei were stained with fluorescence to observe and analyze cell morphology. The staining procedure was as follows: the cell culture medium in the sample wells was aspirated, the samples were washed twice with PBS, and 4% paraformaldehyde was added to cover the cells and fix the cells at room temperature for 20-30 min; PBS was washed 3 times, 5 min each time; 0.2% Triton-100 was added to cover the cells and permeabilized at room temperature for 10 min; PBS was washed 3 times, 5 min each time; then 100 nM rhodamine-labeled phalloidin dye was added to stain F-actin at room temperature in the dark for 30 min; PBS was washed 3 times, 5 min each time; 1 μg mL of PBS was added to cover the cells and permeabilized at room temperature for 10 min. -1DAPI staining solution was used to stain cell nuclei at room temperature in the dark for 10 min; PBS was used to wash the cells three times, 5 min each time; after staining, the cells were removed, inverted onto a glass slide, and fixed with nail polish. Cell morphology was observed under a confocal microscope within 24 h of staining, and the stained images were analyzed using Fiji software. Cell spreading area and semi-quantitative F-actin density were calculated. F-actin density is the F-actin fluorescence intensity per unit cell area: mean fluorescence intensity (Mean) = total fluorescence intensity within a single cell region (IntDen) / area of ​​that cell region (Area). Results showed that radiation-senescent fibroblasts exhibited a flattened morphology (the area of ​​single-radiation-senescent HDF increased by approximately 76% compared to the control group), with expanded F-actin and decreased density. (The F-actin assembly density of single-radiation-senescent HDF decreased by approximately 37% compared to the control group). Figure 3 .

[0117] 6) Cellular Young's modulus determination: The test was conducted using a nanoindenter based on fiber optic interferometry. A probe with a radius of 3 μm was used for indentation, and the hardness of the indentation was 0.017 N / m. -1 Using peak loading mode, nanoindentation testing was performed at 30 μm s⁻¹. -1 The probe was retracted at the same speed after reaching a peak load of 0.03 μN. The mechanical properties of the cells, particularly Young's modulus, were analyzed using Hertzian contact mechanics. Indentation curve fitting was performed using DataViewer V2.5.2 (Optics11 Life) within the linear region of the loading curve, requiring R... 2 > 0.99. The results showed that the Young's modulus of radiation-senescent fibroblasts decreased significantly, reference Figure 4 .

[0118] 7) Collagen shrinkage assay: Collect cells and resuspend them in cell culture medium, ensuring a cell density of 4 × 10⁻⁶ cells / mL. 6 / mL, prepare collagen gel working solution according to the kit instructions. Example: 24 wells, 9.54 mL collagen solution, 2.46 mL PBS, 340 μL neutralization solution (prepare proportionally as needed). Then mix cell suspension and collagen working solution at a ratio of 1:4 to prepare the contraction system. Pipette 500 μL of the contraction system into 24-well plates and incubate at 37 °C, 5% CO2 for 60 min until the collagen solution solidifies. Then add 1 mL of cell culture medium to each well. After 24 h, collect images of the gel. Use Fiji software to count changes in collagen gel size and analyze cell contractility based on the changes in collagen gel size. Results show that radiation-induced senescent fibroblasts significantly lose their ability to contract collagen. Figure 5 .

[0119] 8) Phospho-Myosin light Chain2 (p-MLC) and Myosin light Chain2 (MLC) detection: The expression levels of p-MLC and MLC proteins were determined by Western blotting to analyze the expression level and activity of the motor protein myosin in cells. After extracting and measuring the total protein content, proteins were separated by 12% SDS-PAGE gel. The separated proteins were transferred to PVDF membranes, blocked, and incubated with corresponding antibodies: rabbit polyclonal anti-Phospho-Myosin light Chain2 primary antibody, mouse monoclonal anti-myosin regulatory light chain2 primary antibody, and secondary antibodies of the corresponding species-binding HRP. Finally, the protein bands were exposed under a gel imaging system using ECL chemiluminescence working solution. The results showed that the expression levels of MLC and p-MCL were decreased in radiation-senescent fibroblasts, indicating that the expression level and activity of the motor protein myosin were both reduced. Figure 6 .

[0120] 2. Evaluate the decisive role of the cytoskeleton component F-actin in fibroblast senescence.

[0121] 1) Latrunculin A treatment: Normally cultured primary HDF cells served as the control group (CTRL). As needed, after cell adhesion, 120 nM Latrunculin A (LAT-A) was added to the cells for 24 h to disrupt F-actin polymerization and assembly, forming the test group (LAT-A). After LAT-A treatment of HDF, F-actin was first stained with rhodamine-labeled phalloidin dye, and observed under a confocal microscope within 24 h. This confirmed that LAT-A treatment inhibited F-actin polymerization in fibroblasts, and the intracellular F-actin density decreased after LAT-A treatment. Figure 7 A and B.

[0122] 2) Young's modulus assay: The changes in Young's modulus of cells inhibited by LAT-A-treated primary HDF F-actin assembly were measured using a nanoindenter based on fiber optic interferometry. The results showed that loss of F-actin cytoskeleton integrity affected cytoskeleton tension, leading to a decrease in Young's modulus. Figure 7 C.

[0123] 3) Detection of cell cycle inhibitor P21: Western blotting was used to determine the expression changes of P21, a senescence marker, after LAT-A treatment damaged the primary HDFF-actin cytoskeleton. Results showed that P21 expression significantly increased after LAT-A treatment damaged the F-actin cytoskeleton tension, and significantly decreased after LAT-A removal and F-actin cytoskeleton remodeling. (Reference) Figure 8 .

[0124] 4) Jasplakionlide treatment: Experimental groups: Normally cultured primary HDF served as the control group (CTRL), UVB-irradiated senescent HDF served as the model group (UV), and HDF treated with 20 nM Jasplakionlide (JAK) for 24 h after HDF irradiation served as the test group (UV+Jak). After JAK treatment of HDF, the F-actin backbone was stained with rhodamine-labeled phalloidin dye, and the staining results were observed under a confocal microscope. The results showed that JAK promoted F-actin polymerization in radiation-senescent fibroblasts, which could protect the spindle-shaped morphology of fibroblasts. (Reference) Figure 9 Similarly, Western blotting was used to determine the expression changes of the cell cycle inhibitor P21 after JAK-induced HDF radiation. The results showed a significant decrease in P21 expression. (Reference) Figure 9 B.

[0125] 3. Apply the test substances to photoaged fibroblasts to screen for agents that control cellular senescence.

[0126] 1) Experimental grouping: Normally cultured primary HDF served as the control group (CTRL), UVB-irradiated aged HDF served as the model group (UV), and the test group (163) was supplemented with the test substance bicyclic peptide 163 after irradiation. Bicyclic peptide 163 structure: Cyclo (His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bond bridging Cys & Cys).

[0127] 2) Test substance concentration: 25 ppm.

[0128] 3) Cell morphology protection assay: Primary HDF cells were sparsely seeded in 96-well glass-bottomed cell culture plates and cultured for 24 h before UVB irradiation. After irradiation, the test group was supplemented with medium containing bicyclic peptide 163, while the control and model groups were supplemented with fresh medium. After culturing for another 24 h, F-actin was stained with Alexa Fluor 488-labeled phalloidin and observed under an inverted fluorescence microscope. The results showed that the addition of bicyclic peptide 163 increased the F-actin assembly density of the skeletal framework in senescent HDF cells by approximately 50%, protected F-actin assembly, and maintained the elongated spindle-shaped morphology of cells (the cell aspect ratio was restored to approximately 88% compared to the control group HDF). Figure 10 .

[0129] 4) Detection of cell cycle inhibitor P21: The expression of cell cycle inhibitor P21, a cellular senescence indicator, was measured using Western blotting. Results showed that bicyclic peptide 163 significantly downregulated P21 protein expression. (Reference) Figure 11 .

[0130] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0131] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for evaluating the senescence state of dermal fibroblasts, characterized in that, The method includes: The assembly density of F-actin in dermal fibroblasts was measured and compared with that of a control. If the assembly density is lower than that of the control, the dermal fibroblasts are evaluated as senescent cells or senescent cells. The control group included the assembly density of F-actin in dermal fibroblasts that were definitively identified as non-senescent. Optionally, the unsenescent dermal fibroblasts include young, un-irradiated primary dermal fibroblasts.

2. The method for evaluating the senescence state of dermal fibroblasts as described in claim 1, characterized in that, The method further includes one or more of the following determinations: 1) Determine the cell cycle inhibitory protein P21 in dermal fibroblasts; 2) Determine the Young's modulus of dermal fibroblasts; 3) β-galactosidase staining; 4) Measure collagen contraction in dermal fibroblasts; and, 5) Cell spindle-shaped morphology.

3. A method for evaluating the effect of an analyte on inhibiting and / or improving dermal fibroblast senescence, characterized in that, The method includes: The test substance was mixed with senescent dermal fibroblasts and incubated. The assembly density of F-actin and changes in cell morphology in dermal fibroblasts were measured before and after treatment.

4. The method for evaluating the effect of the analyte on inhibiting and / or improving dermal fibroblast senescence as described in claim 3, characterized in that, If the assembly density of F-actin is increased and the spindle-shaped morphology of cells is preserved before and after treatment, then the analyte is determined to have the effect of inhibiting and / or improving dermal fibroblast senescence.

5. The method for evaluating the effect of the analyte on inhibiting and / or improving dermal fibroblast senescence as described in claim 3 or 4, characterized in that, The method further includes one or more of the following determinations: 1) Determine the cell cycle inhibitory protein P21 in dermal fibroblasts; 2) Determine the Young's modulus of dermal fibroblasts; 3) β-galactosidase staining; and, 4) Measure collagen contraction in dermal fibroblasts.

6. The method for evaluating the effect of the analyte on inhibiting and / or improving dermal fibroblast senescence as described in claim 3 or 4, characterized in that, The senescent dermal fibroblasts mentioned are radiation-senescent fibroblasts.

7. The method for evaluating the effect of the analyte on inhibiting and / or improving dermal fibroblast senescence as described in claim 6, characterized in that, The radiation-senescent fibroblasts are fibroblasts obtained by UVB radiation-induced senescence.

8. The method for evaluating the effect of the analyte on inhibiting and / or improving dermal fibroblast senescence as described in claim 7, characterized in that, The method for preparing radiation-senescent fibroblasts includes: Primary dermal fibroblasts with a cell passage number of 6 or less were exposed to ultraviolet radiation at a dose of 120 mJ / cm². 2 Of the UVB mentioned above.

9. Use of substances that maintain F-actin cytoskeleton homeostasis in the preparation of formulations that inhibit and / or improve cellular senescence.

10. A method for screening agents that inhibit and / or improve dermal fibroblast senescence, characterized in that, The method includes: The candidate material was mixed with senescent dermal fibroblasts and incubated. The assembly density of F-actin and changes in cell morphology in dermal fibroblasts were measured before and after treatment.

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