In-vitro model of skin inflammatory aging as well as establishment method and application of in-vitro model

Abstract

The invention relates to an in-vitro model of skin inflammatory aging as well as an establishment method and application thereof, and relates to the technical field of cell models. The model comprises human fibroblasts and an aging inductor of the fibroblasts, and the aging inductor is a culture medium supernatant of human keratinocytes with inflammation. According to the method disclosed by the invention, the inflammatory culture medium of the keratinocytes is used for inducing the senescence of the fibroblasts, so that the correlation between senescence characteristics of the human keratinocytes and the fibroblasts in an inflammatory environment is more truly reflected, and the key biological process of skin inflammatory senescence is more accurately reflected; the application of the cell model in a real living body environment and the accuracy of disease mechanism research are improved.

Description

Technical Field

[0001] The present disclosure relates to the technical field of cell models, and in particular to an in vitro model of skin inflammatory aging and a method for establishing and applying the model. Background Art

[0002] The skin is the largest organ in the human body and has multiple physiological functions such as protection, sensation and regulation. It consists of three layers: epidermis, dermis and subcutaneous tissue. The epidermis is mainly composed of keratinocytes, which are responsible for forming a protective barrier; the dermis mainly contains fibroblasts, which are responsible for synthesizing collagen and other matrix proteins necessary to maintain the structural integrity and normal function of the skin.

[0003] As we age, the body's immune regulation ability declines, resulting in a lower threshold for inflammatory responses. This chronic inflammatory state accelerates the aging process of tissues and organs and is associated with the occurrence of a variety of age-related diseases. In the study of skin aging, inflammation is considered to be an important factor closely related to the aging process. UVB (Ultraviolet Radiation B) irradiation is a commonly used experimental method to simulate the effects of sunlight on the skin, especially to induce inflammatory responses in keratinocytes. These responses include the release of cytokines, increased oxidative stress, and accumulation of cell damage, and are key biomarkers in the aging process.

[0004] Inflammatory aging is a complex process involving multiple cell types and molecular mechanisms, and various methods have been used to study it. CN113564120A discloses an inflammatory aging model based on lipopolysaccharide-induced mouse peritoneal macrophage cell line RAW264.7 cells. This model uses inflammatory factors such as IL-1α, IL-6, TNF-α, as well as aging-related β-galactosidase, generated reactive oxygen species (ROS), and aging-associated heterochromatic chromatin focus (SAHF) as detection indicators. However, the limitation of this method is that only a single macrophage type is used for research, which fails to fully simulate multicellular interactions and lacks a direct connection between the release of inflammatory factors and cell aging markers. In addition, the biological differences between the use of mouse cells and human cells may also limit the clinical relevance of the model.

[0005] CN111088247A discloses a skin ultraviolet damaged cell model, a method for establishing the model and its application, wherein the method for establishing the skin ultraviolet damaged cell model includes three steps: culturing epidermal cells, damaging epidermal cells by ultraviolet radiation and cell detection. Although this method has low modeling cost, is more sensitive to skin ultraviolet damage stress response and can explore skin ultraviolet damage at the molecular biology level, it only studies the effect of UVB radiation on human keratinocytes and fails to establish the connection between the inflammatory response of the epidermis and the aging process of the dermis.

[0006] Fibroblasts are very sensitive to signals from the epidermis, which affect the aging and repair process of the skin by regulating their synthetic activities. Although UVB irradiation has been widely used to study the effects of environmental factors on the skin, the above and similar studies mainly focus on the direct response of a single cell itself. The cell model is far from the actual human skin microenvironment, and does not truly reflect the correlation between the aging characteristics of human keratinocytes and fibroblasts under an inflammatory environment, and cannot accurately reflect the key biological processes of inflammatory aging of the skin. Summary of the invention

[0007] The purpose of the present disclosure is to provide a cell model that can simulate and study the association between human skin epidermal inflammation and dermal fibroblast aging characteristics. The specific scheme is as follows: An in vitro model of skin inflammatory aging comprises human fibroblasts and a senescence inducer of the fibroblasts, wherein the senescence inducer is a culture supernatant of human keratinocytes with inflammation.

[0008] Preferably, the method of inducing inflammation in the keratinocytes comprises irradiation, chemical induction, gene editing or a combination thereof.

[0009] More preferably, the method of inducing inflammation in the keratinocytes is UVB irradiation.

[0010] Another object of the present disclosure is to provide a method for establishing a cell model capable of simulating and studying the association between human skin epidermal inflammation and dermal fibroblast aging characteristics, the specific scheme is as follows: A method for establishing an in vitro model of skin inflammatory aging comprises the following steps: S1. Cultivating human keratinocytes, inducing inflammation in the keratinocytes, and obtaining a culture supernatant of the keratinocytes having inflammation; S2. culturing human fibroblasts with the culture medium supernatant in step S1 to induce senescence of the fibroblasts; S3. Assessing senescence markers in the fibroblasts after induction of senescence.

[0011] Preferably, the method further comprises the step of evaluating inflammatory factors in the keratinocytes having inflammation.

[0012] More preferably, the evaluation of inflammatory factors includes detecting the expression of IL-6, IL-1α, PGE2 and TNF-α, detecting the expression of IL-17RA, HSP90AA1, TRAF6 and IKBKG, and detecting the expression of c-Fos and c-Jun proteins.

[0013] Preferably, the senescence markers include MMP1, MMP3, P21 and P53.

[0014] More preferably, in step S1, the keratinocytes are induced to produce inflammation by UVB irradiation in a PBS buffer, and after the induction, the PBS buffer is replaced with a complete culture medium to culture the keratinocytes.

[0015] Preferably, in step S2, the fibroblasts are cultured in the culture medium supernatant in step S1 for 2 to 10 days.

[0016] Another object of the present disclosure is to apply the aforementioned in vitro model of skin inflammatory aging to the screening of anti-skin inflammatory aging substances.

[0017] Preferably, the screening of anti-skin inflammatory aging substances comprises: A1. Adding an anti-skin inflammatory aging candidate to the culture of the in vitro model of skin inflammatory aging to induce human fibroblasts therein; A2. evaluating senescence markers of the induced fibroblasts; A3. comparing the senescence markers in step A2 and the senescence markers of the fibroblasts induced by the culture without adding the candidate; A4. Screening out the candidate substances having anti-skin inflammatory and anti-aging functions.

[0018] The technical solution claimed in the present disclosure has achieved the following beneficial effects: 1) The inflammatory culture medium of senescent keratinocytes was used to induce fibroblast senescence, which more realistically reflects the correlation between the aging characteristics of human keratinocytes and fibroblasts under an inflammatory environment, and more accurately reflects the key biological processes of inflammatory aging of the skin, improving the application of cell models in real biological environments and the accuracy of disease mechanism research.

[0019] 2) It not only expands the research on inflammation in cell aging, but also provides a new experimental model for studying how inflammation affects the aging of dermal cells through intercellular signaling. This method reveals that the IL-17 pathway is a target for inducing inflammatory aging, which has potential application value in combating inflammation and aging-related diseases.

[0020] 3) Cell models are simple, easy to operate and low-cost. Compared with 3D skin models or human testing, they have the potential to become an ideal experimental platform in cosmetic and pharmaceutical development, and are suitable for large-scale screening of anti-inflammatory aging candidate substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are merely embodiments of the present disclosure, and a person of ordinary skill in the art can obtain other drawings based on the provided drawings without paying any creative work.

[0022] Figure 1 A flowchart of the steps for constructing the cell model disclosed herein.

[0023] Figure 2 The release amounts of inflammatory factors IL-6 (A), IL-1α (B), PGE2 (C) and TNFα (D) of HaCaT cells after UVB irradiation in Example 1.

[0024] Figure 3 The expression levels of IL-17RA (A), HSP90AA1 (B), TRAF6 (C) and IKBKG mRNA (D) in HaCaT cells after UVB irradiation in Example 2.

[0025] Figure 4 The expression levels of c-Fos and c-JUN proteins in HaCaT cells after UVB irradiation in Example 3.

[0026] Figure 5 The relative expression levels of MMP1 (A), MMP3 (B), COL1A1 (C), P21 (D) and P53 (E) mRNA in HFF cells after culturing in inflammatory medium for 2 days in Example 4.

[0027] Figure 6 The relative expression levels of MMP1 (A), MMP3 (B), COL1A1 (C), P21 (D) and P53 (E) mRNA in HFF cells after culturing in inflammatory medium for 4 days in Example 5.

[0028] Figure 7 The relative expression levels of MMP1 (A), MMP3 (B), COL1A1 (C), P21 (D) and P53 (E) mRNA in HFF cells after 6 days of culture in inflammatory medium in Example 6.

[0029] Figure 8 The relative expression levels of MMP1 (A), MMP3 (B), COL1A1 (C), P21 (D) and P53 (E) mRNA in HFF cells after 10 days of culture in inflammatory medium in Example 7.

[0030] Fig. 9Relative mRNA expression levels of MMP1 (A), MMP3 (B) and P53 (C) in HFF cells after 4 days of culture in inflammatory medium and intervention with roxburghii fruit extract in Application Example 1.

[0031] Fig.10 The release amounts of inflammatory factors IL-6 (A), IL-1α (B), PGE2 (C) and TNFα (D) of HFF cells after UVB irradiation in Comparative Example 1.

[0032] Fig.11 The expression levels of IL-17RA (A), HSP90AA1 (B), TRAF6 (C) and IKBKG mRNA (D) in HFF cells after UVB irradiation in Comparative Example 1.

[0033] Fig.12 The relative mRNA expression levels of MMP1 (A), MMP3 (B), P53 (C), COL1A1 (D) and P21 (E) in HFF cells after UVB irradiation in Comparative Example 1.

[0034] Fig.13 The relative mRNA expression levels of MMP1 (A), MMP3 (B), COL1A1 (C), P53 (D) and P21 (E) in HFF cells after the addition of inflammatory inhibitors in the two models in Comparative Example 2. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and beneficial effects of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0036] like Figure 1 As shown, the disclosed scheme mainly includes three steps: culturing and irradiating keratinocytes, establishing a cell model (inflammatory induction of fibroblasts), and evaluating the cell model: 1) Culture and irradiation of keratinocytes Human keratinocytes HaCaT were used. HaCaT cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum. HaCaT cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. After the culture medium was replaced with PBS buffer solution, it was irradiated using a UVB irradiator with a set dose of 10-30 mJ / cm² to induce an inflammatory response. After irradiation, the PBS was discarded and washed twice with fresh PBS, and then a new complete medium was added to continue culturing for 24 hours. The culture medium of keratinocytes after UVB irradiation was collected and used as an inducer of fibroblasts in subsequent experiments.

[0037] 2) Inflammatory induction of fibroblasts Human fibroblasts HFF were cultured in high-glucose DMEM containing 10% fetal bovine serum. After 24 hours, the culture medium was replaced with the inflammation medium collected from step 1 and cultured for 2-10 days to induce senescence of the fibroblasts.

[0038] 3) Model evaluation After establishing the inflammatory aging model, fibroblasts treated with inflammatory medium were collected and biological analysis was performed to evaluate changes in aging-related markers, such as cytokine expression, ROS generation, and other aging markers.

[0039] The technical solution of the present disclosure is further described in detail below through specific embodiments. Example 1

[0040] This example provides a method for preparing an inflammatory medium for inducing senescence of human fibroblasts.

[0041] Inoculation cells: Digest and collect HaCaT cells (human keratinocytes) in the logarithmic growth phase. Count the HaCaT cells and adjust the cell concentration to 2*10 6 The adjusted HaCaT cells were inoculated into a culture dish with a diameter of 10 cm, and 10 ml of culture medium was added to each dish.

[0042] Treat cells: When the cells grow to the logarithmic growth phase and the degree of confluence is about 60%, discard the old culture medium and replace the culture medium with PBS buffer solution. Irradiate the cells under UVB irradiation equipment, and the irradiation dose is set to 30mJ / cm². After irradiation, discard the PBS buffer and wash the cells twice with fresh PBS buffer. Add fresh complete medium and continue to culture for 24 hours. Collect the culture supernatant after 24 hours, and this supernatant is used as an inflammatory culture medium for the next experiment. Take part of the supernatant for inflammatory factor analysis, and use ELISA kits for IL-6, IL-1α, PGE2 and TNF-α for detection, and measure the absorbance value to calculate the relative release of these inflammatory factors.

[0043] The test results of inflammatory factors are as follows Figure 2 As shown, after UVB irradiation, the release of inflammatory factors IL-6, IL-1α, PGE2, and TNFα increased significantly (p<0.001), indicating that HaCaT cells release a large amount of inflammatory factors after UVB irradiation. Example 2

[0044] This example provides a method for preparing an inflammatory medium for inducing senescence of human fibroblasts.

[0045] Inoculation of cells: Digest and collect cells from HaCaT cells in the logarithmic growth phase. Count the HaCaT cells and adjust the cell concentration to 2*10 6 The adjusted HaCaT cells were inoculated into a culture dish with a diameter of 10 cm, and 10 ml of culture medium was added to each dish.

[0046] Treat cells: When cells grow to the logarithmic growth phase and the confluence is about 60%, discard the old culture medium and replace it with PBS buffer solution. Irradiate the cells under UVB irradiation equipment, and the irradiation dose is set to 30mJ / cm². After irradiation, discard the PBS buffer and wash the cells twice with fresh PBS buffer. Add fresh complete medium and continue to culture for 24 hours. Collect the culture supernatant after 24 hours, and this supernatant is used as inflammation medium for the next experiment. Total RNA is extracted from cells in each treatment group using Trizol reagent, and the purity of RNA is determined. cDNA is synthesized using reverse transcriptase (MLV). Using appropriate amount of cDNA as template, specific fragments of IL-17RA, HSP90AA1, TRAF6 and IKBKG genes are amplified in PCR by Taq DNA polymerase. The reaction conditions of RT-PCR were set as follows: pre-denaturation at 94°C for 5 minutes; followed by 40 cycles, each cycle including 94°C for 30 seconds, 57°C for 35 seconds, and 72°C for 35 seconds; and finally extension at 72°C for 10 minutes.

[0047] The RT-PCR results are as follows Figure 3 As shown in the figure, after UVB irradiation, the relative expression of IL-17RA, HSP90AA1, TRAF6 and IKBKG genes increased significantly (p<0.001). These genes are all key genes in the IL-17 signaling pathway, so UVB irradiation may induce HaCaT cells to activate the IL-17 pathway. Activation of the IL-17 pathway can stimulate keratinocytes to release a variety of inflammatory factors, which further recruit and activate neutrophils and other inflammatory cells to amplify the inflammatory response. The IL-17 pathway plays an important role in inflammatory aging by promoting inflammatory responses and the secretion of SASP factors. It is not only a key regulator of inflammatory responses, but also plays a role in cell aging and tissue dysfunction. Example 3

[0048] This example provides a method for preparing an inflammatory medium for inducing senescence of human fibroblasts.

[0049] Inoculation of cells: Digest and collect cells from HaCaT cells in the logarithmic growth phase. Count the HaCaT cells and adjust the cell concentration to 2*10 6 The adjusted HaCaT cells were inoculated into a culture dish with a diameter of 10 cm, and 10 ml of culture medium was added to each dish.

[0050] Treat cells: When HaCaT cells grow to the logarithmic growth phase and the confluence is about 60%, discard the old culture medium and replace it with PBS buffer solution. Irradiate the cells under UVB irradiation equipment, and the irradiation dose is set to 30mJ / cm². After irradiation, discard the PBS buffer and wash the cells twice with fresh PBS buffer. Add fresh complete medium and continue to culture for 24 hours. Collect the culture supernatant after 24 hours, which is used as inflammation medium for the next experiment.

[0051] After harvesting the cells, they were washed twice with cold PBS buffer to remove residual culture medium. Cells were lysed with cell lysis buffer at 4°C to prepare cell lysates for subsequent protein analysis. Protein concentrations in cell lysates were determined using an enhanced BCA protein assay kit (Biyuntian Biotechnology Co., Ltd., Shanghai, China). Equal amounts of protein in each sample were separated by SDS-PAGE to ensure consistency in comparison between samples. Separated proteins were transferred to PVDF membranes for further analysis. Nonspecific binding sites were blocked using TBST solution containing 10% skim milk powder. Target proteins were probed with specific primary antibodies and subsequently labeled with corresponding HRP-conjugated secondary antibodies. Proteins were visualized using Chemistar™ High Sensitivity ECL Western Blotting Substrate. Images were recorded using the Tianneng-5200 Chemiluminescent Imaging System (Tianneng Life Science Co., Ltd., Shanghai, China). Image analysis was performed using Image J software to quantify changes in protein expression.

[0052] The protein expression test results are as follows Figure 4 As shown in the figure, after UVB irradiation, the expression of c-Fos and c-Jun proteins in HaCaT cells increased significantly. c-Fos and c-Jun are two main components of AP-1 (Activator Protein-1) transcription factors. After AP-1 activation, it can promote the expression of a variety of inflammatory factors. Therefore, UVB irradiation can cause inflammatory responses in HaCaT cells by activating AP-1. Example 4

[0053] This example provides a method for establishing an in vitro model of skin inflammatory aging.

[0054] Inoculation cells: Take cells from HFF (human fibroblasts) in the logarithmic growth phase, digest and collect them. Count the HFF cells and adjust the cell concentration to 1.5*10 5 The adjusted HFF cells were seeded in 6-well plates and 2 ml of culture medium was added to each well.

[0055] Treating cells: When HFF cells grow to the logarithmic growth phase and the confluence is about 60%, the old culture medium is discarded. The inflammatory culture medium collected from Example 1 is added and culture is continued for 2 days to induce inflammatory and senescent responses. Total RNA was extracted from cells in each treatment group using Trizol reagent, and the purity of RNA was determined. cDNA was synthesized using reverse transcriptase (MLV). Using an appropriate amount of cDNA as a template, specific fragments of MMP1, MMP3, COL1A1, P21 and P53 genes were amplified in PCR by Taq DNA polymerase. The reaction conditions of RT-PCR were set as: pre-denaturation at 94°C for 5 minutes; then 40 cycles, each cycle including 94°C for 30 seconds, 57°C for 35 seconds, and 72°C for 35 seconds; finally, extension at 72°C for 10 minutes.

[0056] RT-PCR reaction results Figure 5 As shown, after 2 days of induction of HFF cells with inflammatory medium, the mRNA expression of MMP1, MMP3, P21 and P53 in HFF cells increased significantly (p<0.001), while the mRNA expression of COL1A1 (collagen) decreased significantly (p<0.05). These results indicate that inflammatory medium can induce changes in the expression of factors related to aging in HFF cells and promote the aging of HFF cells. Example 5

[0057] This example provides a method for establishing an in vitro model of skin inflammatory aging.

[0058] Inoculation cells: Take cells from HFF cells in the logarithmic growth phase, digest and collect them. Count the HFF cells and adjust the cell concentration to 1.5*10 5 The adjusted HFF cells were seeded in 6-well plates and 2 ml of culture medium was added to each well.

[0059] Treating cells: When HFF cells grow to the logarithmic growth phase and the confluence is about 60%, the old culture medium is discarded. The inflammatory culture medium collected from Example 1 is added and culture is continued for 4 days to induce inflammation and senescence response. Total RNA is extracted from cells in each treatment group using Trizol reagent, and the purity of RNA is determined. cDNA is synthesized using reverse transcriptase (MLV). Using an appropriate amount of cDNA as a template, specific fragments of MMP1, MMP3, COL1A1, P21 and P53 genes are amplified in PCR by Taq DNA polymerase. The reaction conditions of RT-PCR are set as: pre-denaturation at 94°C for 5 minutes; then 40 cycles, each cycle including 94°C for 30 seconds, 57°C for 35 seconds, and 72°C for 35 seconds; finally, extension at 72°C for 10 minutes.

[0060] RT-PCR reaction results Figure 6As shown: After 4 days of induction of HFF cells with inflammatory medium, the mRNA expression of MMP1, MMP3, P21 and P53 in HFF cells increased significantly (p<0.001), while the mRNA expression of COL1A1 decreased significantly (p<0.01). These results indicate that inflammatory medium can induce changes in the expression of factors related to aging in HFF cells and promote the aging of HFF cells. Example 6

[0061] This example provides a method for establishing an in vitro model of skin inflammatory aging.

[0062] Inoculation cells: Take cells from HFF cells in the logarithmic growth phase, digest and collect them. Count the HFF cells and adjust the cell concentration to 1.5*10 5 The adjusted HFF cells were seeded in 6-well plates and 2 ml of culture medium was added to each well.

[0063] Treating cells: When HFF cells grow to the logarithmic growth phase and the confluence is about 60%, the old culture medium is discarded. The inflammatory culture medium collected from Example 1 is added and culture is continued for 6 days to induce inflammation and senescence responses. Total RNA is extracted from cells in each treatment group using Trizol reagent, and the purity of RNA is determined. cDNA is synthesized using reverse transcriptase (MLV). Using an appropriate amount of cDNA as a template, specific fragments of MMP1, MMP3, COL1A1, P21 and P53 genes are amplified in PCR by Taq DNA polymerase. The reaction conditions of RT-PCR are set as: pre-denaturation at 94°C for 5 minutes; then 40 cycles, each cycle including 94°C for 30 seconds, 57°C for 35 seconds, and 72°C for 35 seconds; finally, extension at 72°C for 10 minutes.

[0064] RT-PCR reaction results Figure 7 As shown: After 6 days of induction of HFF cells with inflammatory medium, the mRNA expression of MMP1, MMP3, P21 and P53 in HFF cells increased significantly (p<0.001), while the mRNA expression of COL1A1 decreased significantly (p<0.01). These results indicate that inflammatory medium can induce changes in the expression of factors related to aging in HFF cells and promote the aging of HFF cells. Example 7

[0065] This example provides a method for establishing an in vitro model of skin inflammatory aging.

[0066] Inoculation cells: Take cells from HFF (human fibroblasts) in the logarithmic growth phase, digest and collect them. Count the HFF cells and adjust the cell concentration to 1.5*10 5The adjusted HFF cells were seeded in 6-well plates and 2 ml of culture medium was added to each well.

[0067] Treating cells: When HFF cells grow to the logarithmic growth phase and the confluence is about 60%, the old culture medium is discarded. The inflammatory culture medium collected from Example 1 is added and cultured for 10 days to induce inflammation and senescence responses. Total RNA was extracted from cells in each treatment group using Trizol reagent, and the purity of RNA was determined. cDNA was synthesized using reverse transcriptase (MLV). Using an appropriate amount of cDNA as a template, specific fragments of MMP1, MMP3, COL1A1, P21 and P53 genes were amplified in PCR by Taq DNA polymerase. The reaction conditions of RT-PCR were set as: pre-denaturation at 94°C for 5 minutes; then 40 cycles, each cycle including 94°C for 30 seconds, 57°C for 35 seconds, and 72°C for 35 seconds; and finally extension at 72°C for 10 minutes.

[0068] RT-PCR reaction results Figure 8 As shown: After 10 days of induction of HFF cells with inflammatory medium, the mRNA expression of MMP1 and MMP3 in HFF cells increased significantly (p<0.001), while the mRNA expression of COL1A1, P21 and P53 decreased significantly (p<0.01). These results indicate that inflammatory medium can induce changes in the expression of factors related to aging in HFF cells and promote the aging of HFF cells.

[0069] It can be seen from Examples 4-7 that as the number of days of inflammatory culture medium culture increases, the aging markers MMP1, MMP3, P21 and P53 gradually increase, and COL1A1 gradually decreases, indicating that the inflammatory culture medium is directly related to the cell senescence of fibroblasts, indicating that the in vitro simulation method of inflammatory aging has been successfully established. Application Example 1

[0070] This application example uses Rosa roxburghii fruit as an example to illustrate the application of the in vitro model of skin inflammatory aging of the present application.

[0071] Preparation of inflammation culture medium: Digest and collect HaCaT cells in the logarithmic growth phase. Count the HaCaT cells and adjust the cell concentration to 2*10 6 The adjusted HaCaT cells were inoculated into a culture dish with a diameter of 10 cm, and 10 ml of culture medium was added to each dish.

[0072] Treat cells: When HaCaT cells grow to the logarithmic growth phase and the confluence is about 60%, discard the old culture medium and replace it with PBS buffer solution. Irradiate HaCaT cells under UVB irradiation equipment, and the irradiation dose is set to 30mJ / cm². After irradiation, discard the PBS buffer and wash the cells twice with fresh PBS buffer. Add fresh complete medium and continue to culture for 24 hours. Collect the culture supernatant after 24 hours, which is used as inflammation medium for the next experiment.

[0073] Establishment of inflammatory aging model: HFF cells in the logarithmic growth phase were digested and collected. After cell counting, the cell concentration was adjusted to 1.5*10 5 cells / ml. The adjusted HFF cells were evenly inoculated into each well of a 6-well plate, and 2 ml of culture medium was added to each well. When the cells grew to the logarithmic growth phase and the confluence reached about 60%, the old culture medium was removed. The inflammatory medium prepared in the first step was added and cultured for 4 days, followed by the addition of 200 μg / ml of roxburghii fruit extract, and the culture was continued for 4 days to induce inflammatory response and simulate aging.

[0074] Total RNA was extracted from the treated cells using Trizol reagent and its purity was tested. cDNA was synthesized from the extracted RNA using reverse transcriptase (MLV). PCR amplification was performed using Taq DNA polymerase with an appropriate amount of cDNA as a template, specifically targeting the expression of MMP1, MMP3, and P53 genes. The RT-PCR reaction conditions were set as follows: initial denaturation at 94°C for 5 minutes; followed by 40 cycles, each cycle including 94°C for 30 seconds, 57°C for 35 seconds, and 72°C for 35 seconds; the last extension step was performed at 72°C for 10 minutes.

[0075] RT-PCR reaction results Fig. 9 As shown, after 4 days of induction of HFF cells with inflammatory medium, the mRNA expression of MMP1 and MMP3 in HFF cells was significantly increased (p<0.001), while the mRNA expression of P53 was significantly decreased (p<0.001). After adding 200 μg / ml of roxburghii fruit extract, the mRNA expression of MMP1, MMP3 and P53 in HFF cells was significantly decreased (p<0.001). These results show that roxburghii fruit extract has excellent anti-inflammatory and aging effects and is suitable for use as a raw material in skin care products. Comparative Example 1

[0076] In this comparative example, the culture medium from senescent keratinocytes was not used, but HFF cells were directly irradiated with UVB to detect inflammatory factors and senescence markers.

[0077] Inoculation cells: Take cells from HFF cells in the logarithmic growth phase, digest and collect them. Count the HFF cells and adjust the cell concentration to 1.5*10 5 The adjusted HFF cells were seeded in 6-well plates and 2 ml of culture medium was added to each well.

[0078] Treat cells: When HFF cells grow to the logarithmic growth phase and the confluence is about 60%, discard the old culture medium and replace the culture medium with PBS buffer solution. Irradiate the cells under UVB irradiation equipment, and the irradiation dose is set to 30 mJ / cm². After irradiation, discard PBS and wash the cells twice with fresh PBS. Add fresh complete medium and continue to culture for 24 hours. Collect the culture supernatant after 24 hours and detect it using ELISA kits for IL-6, IL-1α, PGE2 and TNF-α, and measure the absorbance value to calculate the relative release of these inflammatory factors.

[0079] like Fig.10 As shown in the data, after UVB irradiation, the release of the inflammatory factor IL-6 increased significantly (p<0.01), but the release of IL-1α, PGE2, and TNFα did not change significantly, which indicates that HFF cells will release some inflammatory factors after UVB irradiation, but will not release a large amount of inflammatory factors.

[0080] PCR experiment: When the cells grew to the logarithmic growth phase and the confluence was about 60%, the old culture medium was discarded. The cells were irradiated under UVB irradiation equipment, and the irradiation dose was set to 30 mJ / cm². After irradiation, PBS was discarded and the cells were washed twice with fresh PBS. Fresh complete medium was added and cultured for another 24 hours. Total RNA was extracted from the cells of each treatment group using Trizol reagent, and the purity of RNA was determined. cDNA was synthesized using reverse transcriptase (MLV). Using an appropriate amount of cDNA as a template, specific fragments of IL-17, HSP90AA1, TRAF6, IKBKG, MMP1, MMP3, COL1A1, P21 and P53 genes were amplified in PCR by Taq DNA polymerase. The reaction conditions of RT-PCR were set as follows: pre-denaturation at 94℃ for 5 minutes; then 40 cycles, each cycle including 94℃ for 30 seconds, 57℃ for 35 seconds, and 72℃ for 35 seconds; and finally extension at 72℃ for 10 minutes.

[0081] like Fig.11 As shown, there was no significant change in the gene expression levels of IL-17RA, HSP90AA1, TRAF6, and IKBKG after UVB irradiation, indicating that direct UVB irradiation of HFF cells could not promote the IL-17 pathway to produce corresponding inflammatory responses and further cause cell changes.

[0082] like Fig.12 As shown, UVB directly induced HFF cells, and the mRNA expression of MMP1, MMP3, P21 and P53 in HFF cells increased significantly (p<0.001), while the mRNA expression of COL1A1 decreased significantly (p<0.05). These results show that UVB can induce changes in the expression of factors related to aging in HFF cells and promote the aging of HFF cells, but the above experiments show that UVB-induced HFF cell aging may not be caused by inflammatory response, but other reasons. Comparative Example 2

[0083] This comparative example studies the characterization of aging-related genes in each model after adding inflammatory inhibitors to the UVB model and the inflammatory culture medium model.

[0084] Inoculation cells: Take cells from HFF cells in the logarithmic growth phase, digest and collect them. Count the cells and adjust the cell concentration to 1.5*10 5 The adjusted HFF cells were seeded in 6-well plates and 2 ml of culture medium was added to each well.

[0085] PCR experiment: When the cells grow to the logarithmic growth phase and the confluence is about 60%, discard the old culture medium.

[0086] 1) Establishing UVB model: UVB group: cells were irradiated under UVB irradiation equipment, and the irradiation dose was set to 30 mJ / cm². After irradiation, PBS was discarded and cells were washed twice with fresh PBS; UVB plus inhibitor group: the inhibitor IL-17A inhibitor 2 was added after the cells were irradiated; 2) Establishing the Inflammatory Culture Medium Model: Inflammation medium group: the inflammation medium collected from Example 1 was added and culture was continued for 2 days to induce inflammation and senescence response.

[0087] Inflammation medium plus inhibitor group: The inflammation medium collected from Example 1 was added and the inhibitor IL-17A inhibitor 2 was added accordingly, and the culture was continued for 2 days to induce inflammation and senescence response.

[0088] Total RNA was extracted from cells in each treatment group using Trizol reagent, and the purity of RNA was determined. cDNA was synthesized using reverse transcriptase (MLV). Appropriate amounts of cDNA were used as templates to amplify specific fragments of MMP1, MMP3, COL1A1, P21, and P53 genes in PCR using Taq DNA polymerase. The reaction conditions for RT-PCR were set as follows: pre-denaturation at 94°C for 5 minutes; followed by 40 cycles, each cycle including 94°C for 30 seconds, 57°C for 35 seconds, and 72°C for 35 seconds; and finally extension at 72°C for 10 minutes.

[0089] The results are as follows Fig.13 As shown, both direct UVB irradiation and inflammatory medium induction will cause changes in the corresponding aging factors of fibroblasts and induce cell senescence. However, after the addition of inflammatory inhibitors, the aging factors induced by the inflammatory medium will return to normal levels, while the UVB-induced cell senescence has no significant changes after the addition of inflammatory inhibitors. Therefore, UVB-induced senescence may be caused by other reasons, and the inflammatory medium model in the present disclosure is specific due to inflammatory aging induced by inflammation.

[0090] In the scheme disclosed herein, in addition to using UVB irradiation in the embodiment, other types of radiation sources may also be used to induce keratinocyte aging, such as UVA or visible light, which can also induce inflammatory responses in keratinocytes. In addition, chemical inducers such as hydrogen peroxide (H2O2) or other oxidants may also be used to induce keratinocyte aging, and the chemical inducers may be directly added to the culture medium of keratinocytes. Gene editing techniques such as CRISPR or other gene editing tools may also be used to directly modify the gene expression of keratinocytes or fibroblasts to simulate inflammatory responses or aging processes. This method can very accurately regulate specific inflammation or aging-related genes, providing more possibilities for controlling variables. In addition to using fibroblasts, other types of skin cells such as melanocytes or immune cells may also be considered to study the interaction between inflammation and aging.

[0091] Most cell aging models in the prior art focus on directly inducing aging through chemicals or genetic manipulation, lack the simulation of environmental factors (such as inflammation caused by UV radiation), or only focus on a single cell type, such as independent keratinocyte or fibroblast research, and fail to simulate the interaction between cells. The cell model established in the present disclosure uses UVB to irradiate keratinocytes, simulates the effect of sunlight on the epidermis of the skin, induces skin inflammation, and then uses this culture medium to induce dermal fibroblasts, simulating the skin microenvironment, which is closer to the aging mechanism in the natural environment and closer to the interrelationship of human skin tissue. It not only expands the research on inflammation in cell aging, but also provides a new experimental model for studying how inflammation affects the aging of dermal cells through intercellular signaling. This method may also reveal new therapeutic targets and has potential application value in combating inflammation and aging-related diseases. In addition, the model disclosed in the present disclosure has the characteristics of being simple, easy to operate and low cost. Compared with 3D skin models or human tests, it has the potential to become an ideal experimental platform in the development of cosmetics and medicines, and is suitable for large-scale screening of candidate substances for anti-inflammatory aging.

[0092] The embodiments and application examples described above are merely illustrative descriptions of the present disclosure and are not intended to limit the scope of the present disclosure. Without departing from the design spirit of the present disclosure, various modifications and improvements made to the technical solutions of the present disclosure by ordinary technicians in this field should all fall within the protection scope determined by the present disclosure.

Claims

1. An in vitro model of skin inflammatory aging, characterized in that: The invention comprises human fibroblasts and a senescence inducer of the fibroblasts, wherein the senescence inducer is a culture supernatant of human keratinocytes with inflammation.

2. The in vitro model of skin inflammatory aging according to claim 1, characterized in that The methods of inducing inflammation in the keratinocytes include irradiation, chemical induction, gene editing or a combination thereof.

3. The in vitro model of skin inflammatory aging according to claim 2, characterized in that: The method of inducing inflammation in the keratinocytes is UVB irradiation.

4. A method for establishing an in vitro model of skin inflammatory aging according to claim 1, characterized in that: The following steps are involved: S1. Cultivating human keratinocytes, inducing inflammation in the keratinocytes, and obtaining a culture supernatant of the keratinocytes having inflammation; S2. culturing human fibroblasts with the culture medium supernatant in step S1 to induce senescence of the fibroblasts; S3. Assessing senescence markers in the fibroblasts after induction of senescence.

5. The method for establishing an in vitro model of skin inflammatory aging according to claim 4, characterized in that: Also included is the step of evaluating inflammatory factors in said keratinocytes having inflammation.

6. The method for establishing an in vitro model of skin inflammatory aging according to claim 5, characterized in that: The evaluation of inflammatory factors includes detecting the expression of IL-6, IL-1α, PGE2 and TNF-α, detecting the expression of IL-17RA, HSP90AA1, TRAF6 and IKBKG, and detecting the expression of c-Fos and c-Jun proteins; The senescence markers include MMP1, MMP3, P21 and P53.

7. The method for establishing an in vitro model of skin inflammatory aging according to claim 4, characterized in that: In step S1, the keratinocytes are induced to produce inflammation by UVB irradiation in a PBS buffer, and after the induction, the PBS buffer is replaced with a complete culture medium to culture the keratinocytes.

8. The method for establishing an in vitro model of skin inflammatory aging according to claim 4, characterized in that: In step S2, the fibroblasts are cultured in the culture medium supernatant in step S1 for 2 to 10 days.

9. Use of an in vitro model of skin inflammatory aging according to any one of claims 1 to 3, characterized in that: The in vitro model of skin aging is used to screen anti-skin inflammatory aging substances.

10. Use of the in vitro model of skin inflammatory aging according to claim 9, characterized in that: The screening of anti-skin inflammatory aging substances includes: A1. adding an anti-skin inflammatory aging candidate to the culture of the in vitro model of skin aging to induce human fibroblasts therein; A2. evaluating senescence markers of the induced fibroblasts; A3. comparing the senescence markers in step A2 and the senescence markers of the fibroblasts induced by the culture without adding the candidate; A4. Screening out the candidate substances having anti-skin inflammatory and anti-aging functions.

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