Evaluation method for high-temperature thermal damage resistance efficacy of cosmetics

Abstract

The invention discloses a method for evaluating the high-temperature thermal damage resistance effect of cosmetics, and belongs to the technical field of research and evaluation of the effects of cosmetics and raw materials thereof. The invention relates to a method for evaluating the high-temperature thermal injury resistance effect of cosmetics, which comprises the following steps of: performing in-vitro culture by using a skin explant as an experimental model, performing slicing, dyeing, qPCR (quantitative polymerase chain reaction) and the like after high-temperature injury modeling, and detecting the skin morphological structure, extracellular matrix related indexes and heat shock protein gene expression. The method has the advantages of high authenticity and strong intuition, can directly reflect the high-temperature thermal damage resistance effect of the product or the raw material on the skin, has a wide application range, and is not only suitable for cosmetic finished products and raw materials, but also suitable for samples with poor dissolvability such as oil dissolvability and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic efficacy research and evaluation, and in particular to a method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage. Background Art

[0002] The skin is the largest organ in the human body and plays an important role as a barrier and protective layer on the body's surface. The skin not only protects the body's various tissues and organs from external physical, chemical, and other harmful factors, but also prevents the loss of nutrients such as water and electrolytes, thereby maintaining the body's internal homeostasis. Long-term exposure to high temperatures can lead to skin aging, a phenomenon known as heat aging. High temperatures can cause skin moisture loss, dryness, scaling, and decreased skin elasticity. Dehydrated skin is more prone to fine lines and wrinkles. High temperatures can also damage the sebum membrane, weakening the skin's barrier function and making it more vulnerable to external harmful substances. In addition, heat aging can trigger oxidative stress responses in skin cells, generating large amounts of free radicals, causing DNA damage, and leading to excessive degradation of collagen and elastin.

[0003] When the skin is exposed to temperatures above its physiological temperature, it can cause thermal damage. The extent of this damage depends on factors such as the duration of exposure, the temperature, and individual variability. Phenotypically, this can manifest as inflammatory reactions such as pain, erythema, and edema. In severe cases, it can lead to tissue necrosis and deep burns. High temperatures disrupt the structure of the epidermal stratum corneum and damage epidermal cells, leading to cell shedding, increased water loss, and the penetration of external irritants, thus affecting the skin's appearance and normal physiological function. High temperatures can further damage the dermis, for example, by denaturing and degrading dermal collagen, affecting collagen assembly, leading to decreased skin elasticity and wrinkle formation. Furthermore, high temperatures can cause skin inflammation and alter protein and cell structure, leading to heat shock and cell death. High temperatures induce the expression of heat shock proteins, which help cells resist heat stress and maintain protein homeostasis. Furthermore, high temperatures increase the production of reactive oxygen species (ROS), causing oxidative stress and cell damage. Increased temperature can stimulate skin cells to release inflammatory factors (such as IL-1β, TNF-α), triggering an inflammatory response. The inflammatory response attracts immune cells (such as neutrophils and macrophages) to infiltrate, further exacerbating tissue damage.

[0004] Thermal damage to the skin can be effectively prevented by reducing exposure to high temperatures and using sunscreen or heat-resistant skincare products. However, current evaluation methods for cosmetics' resistance to skin damage primarily focus on photodamage, such as UV damage, blue light damage, or chemical damage. There is still a lack of testing methods and approaches to assess the resistance of cosmetics and their ingredients to thermal damage.

[0005] The patent with publication number CN118853809A discloses a method for evaluating the efficacy of sunscreen cosmetics against photothermal damage and its application. The patent uses a 3D epidermal model in combination with a human primary fibroblast co-culture model and a 3D full-thickness skin model to screen and evaluate the efficacy of sunscreen cosmetics against photothermal damage based on six indicators, including heat shock gene detection, PGE2 detection, ROS detection, and detection of COL17 protein at the epidermal junction. However, the patent uses a solar simulator (UVA+UVB) for photothermal modeling, and the photothermal damage caused to the skin is mainly ultraviolet damage. In the evaluation of the resistance of cosmetics and their raw materials to high-temperature thermal damage, this method cannot exclude the influence of ultraviolet rays themselves on the evaluation results. Moreover, the model used in this method is quite different from real human skin, and the authenticity, reliability, and scientificity of its evaluation results are poor. In addition, the patent is for the efficacy evaluation of sunscreen cosmetics and cannot be applied to other cosmetics and their raw materials, and the evaluation time is long, requiring at least 4 weeks. Summary of the Invention

[0006] To address the existing problems of the lack of in vitro alternative methods for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage, the present invention provides a method for evaluating the resistance of cosmetics to high-temperature thermal damage. The present invention uses skin explants as an experimental model for in vitro culture. After high-temperature damage modeling, the skin is sliced, stained, and subjected to qPCR and other treatments to detect skin morphology, extracellular matrix-related indicators, and heat shock protein gene expression. This method is highly authentic and intuitive, can directly reflect the efficacy of products or raw materials in resisting high-temperature thermal damage on the skin, and has a wide range of applications. It is not only applicable to finished cosmetic products and raw materials, but also to samples with poor solubility, such as oil-soluble substances.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage comprises the following steps: S1: After disinfecting the skin explant, clean the blood and adhesions on the skin surface with a balanced salt solution, peel off the subcutaneous tissue, and divide the skin tissue into equal-sized skin slices. Place them with the dermis facing down and the epidermis facing up on a gelatin sponge pre-soaked with culture medium, add culture medium, and culture in vitro; S2: The skin explants cultured in vitro were divided into a normal control group, a model control group, and a sample group. After the samples were given and cultured for 24 hours, the model control group and the sample group were subjected to high temperature modeling, and the samples were collected after modeling; S3: The skin explants of each group were cleaned, fixed, embedded, sliced, and then stained to detect the skin morphology and extracellular matrix indicators.

[0008] Preferably, the skin explant in S1 is washed 2-3 times with a balanced salt solution and 1-2 ml of culture medium is added.

[0009] Preferably, the culture medium of the samples in S1 is aspirated and discarded, and the normal control group and the model control group are replaced with 1.5 mL of fresh culture medium, and the sample group is directly replaced with fresh culture medium containing the sample or replaced with fresh culture medium and 45-50 μL of the sample is dripped or smeared on the surface of the skin explant.

[0010] Preferably, the sample concentration is 0.01%-5% or 0.1%-5%.

[0011] Preferably, the sample group is administered by liquid phase administration or surface application.

[0012] More preferably, for samples administered in liquid form, the medium is replaced with 1.5 mL of fresh culture medium containing 0.01%-5% sample; for raw material samples administered by surface application, the medium is replaced with 1.5 mL of fresh culture medium, and 45-50 μL of 0.1%-5% sample is dripped or smeared on the surface of the skin explant; for finished product samples administered by surface application, the medium is replaced with 1.5 mL of fresh culture medium, and 45-50 μL of the original sample solution is dripped or smeared on the surface of the skin explant.

[0013] Skin explants refer to donated or discarded human skin tissue obtained from clinical settings, which is cultured in a specific way to form an efficacy testing model. "Skin explants" are completely consistent with the structure of real in vivo skin, including the stratum corneum, epidermis, DEJ layer, dermis, immune cells, etc. The homology between the zebrafish and human genomes is only 87%, and the skin structures of zebrafish and humans are quite different; 2D cell models cannot truly evaluate the results from a tissue level; 3D reconstructed skin models are artificially constructed multi-layer cells, and their structure and function are quite different from real human skin. Therefore, the present invention uses skin explants as a model.

[0014] In vitro culture of skin explants: After ethical review and informed consent from the skin sample donor or their guardian, obtain donated or discarded skin explants from the hospital. Human foreskin-derived skin explants are preferred for experiments, but skin explants from other locations can also be used in this manner. Ensure a clean laboratory environment and perform necessary disinfection. Fresh skin explants obtained from the clinic are placed in physiological saline and transported quickly and at low temperature to the laboratory for experiments.

[0015] Disinfect the skin explant in alcohol, then use a balanced salt solution to wash away blood and adhesions on the skin surface. Carefully peel the subcutaneous tissue using surgical scissors and forceps, and divide the skin tissue into equal-sized pieces. Cut the absorbable gelatin sponge into equal sizes and place it in a cell culture plate. Add culture medium and allow the sponge to absorb until completely saturated. Place the divided skin explant with the dermis facing down and the epidermis facing up on a gelatin sponge pre-soaked with culture medium. Add an appropriate amount of culture medium to each well to maintain an air-liquid culture environment for the skin explant.

[0016] Preferably, in said S2, the step of in vitro culturing is: placing the skin explant in an incubator at 35-37° C. and containing 4-6% CO 2 for culturing.

[0017] More preferably, the skin explants are cultured in an incubator at 37°C containing 5% CO2.

[0018] More preferably, the model control group and the sample group in S2 are placed in a high-temperature incubator at 55-65° C. for 2-4 hours for high-temperature modeling.

[0019] More preferably, the model control group and the sample group are placed in a 65°C high temperature incubator for 3 hours to form the model.

[0020] Preferably, in S3, the skin explant is subjected to one of the following steps: paraffin embedding and sectioning, OCT embedding and sectioning, extraction of skin tissue mRNA, and reverse transcription.

[0021] Preferably, in S3, the skin explant is stained with hematoxylin-eosin or Masson's stain.

[0022] Preferably, in S3, the skin morphology and extracellular matrix indicators are skin tissue morphology, collagen fiber density, collagen fiber and elastic fiber signal intensity, and heat shock protein HSP70 gene expression indicator.

[0023] Preferably, in S3, the skin morphology and extracellular matrix indicators are photographed using a biological microscope or a two-photon microscope or observed by qPCR.

[0024] More preferably, for skin explants undergoing hematoxylin-eosin staining, fixation, paraffin embedding, sectioning, hematoxylin-eosin staining, and biological microscopy observation are performed. For skin explants used for collagen fiber density detection, fixation, paraffin embedding, sectioning, Masson staining, and biological microscopy observation are performed. For skin explants used for collagen fiber and elastic fiber expression detection, fixation, dehydration, OCT embedding, cryosectioning, and two-photon microscopy observation are performed. For skin explants used for heat shock protein HSP70 gene expression detection, skin tissue mRNA is extracted and reverse transcribed, and qPCR analysis is performed.

[0025] Preferably, in S3, Image J software or SPSS 26.0 software is used to analyze the skin morphology and extracellular matrix indicators.

[0026] More preferably, the collagen fiber density of Masson-stained sections is analyzed using Image J software, the collagen fiber signal intensity and elastic fiber signal intensity detected by two-photon microscopy are analyzed using Image J software, and statistical analysis is performed using SPSS26.0 software. The statistical analysis results of skin tissue morphology, collagen fiber density, collagen fiber and elastic fiber signal intensity, and heat shock protein HSP70 gene expression indicators are used to comprehensively evaluate the sample's ability to resist high-temperature thermal damage.

[0027] Furthermore, to examine the expression of the heat shock protein HSP70 gene, mRNA was extracted from skin tissue after high-temperature exposure. Reverse transcription and qPCR were performed to measure HSP70 gene expression. SPSS 26.0 software was used to statistically analyze the relative expression of the HSP70 gene.

[0028] Therefore, the present invention has the following beneficial effects: (1) Skin explants, i.e., real human skin tissues, are selected as the evaluation model, and the evaluation results are true and reliable. (2) The evaluation of the efficacy of skin explants in resisting high-temperature thermal damage only takes 2-4 days, while human trials and 3D reconstructed skin trials require at least 4 weeks, which significantly shortens the evaluation cycle. (3) It complies with the 3R principle of animal protection and welfare: experiments are conducted using clinically donated or discarded skin tissues obtained after ethical review and informed consent from patients, and no animal experiments are involved. (4) The experimental results are visualized and easy to understand: the morphological structure of the dermis and epidermis of the skin explants and the damaging effects of high temperature on the skin can be directly observed by hematoxylin-eosin staining, the density of collagen fibers in the skin explants can be directly observed by Masson staining, and the expression of collagen fibers and elastic fibers in the skin explants (signal strength) can be directly observed by two-photon microscopy imaging. (5) Wide range of applications and high tolerance: Samples can be administered in liquid phase or applied to the surface. It is applicable not only to water-soluble samples but also to water-insoluble samples such as oil-soluble samples, and is particularly suitable for spreadable samples such as creams and lotions. Zebrafish and cell-based assays, on the other hand, often have low tolerance for high-concentration samples and are only applicable to water-soluble samples. (6) The present invention uses two-photon microscopy technology to achieve simultaneous imaging of collagen fibers and elastic fibers in skin explants, eliminating the need for complex steps such as fluorescent staining, significantly shortening the evaluation cycle and simplifying the experimental process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The present invention is a flowchart of a method for evaluating the resistance of cosmetics to high-temperature thermal damage.

[0030] Figure 2 This is the morphological structure of the dermis and epidermis of each group of skin explants after high temperature damage in Example 1.

[0031] Figure 3 3 is a graph of collagen fiber density in the dermis of skin explants of each group in Example 2.

[0032] Figure 4 Statistical graph of collagen fiber density in the dermis of skin explants of each group in Example 2.

[0033] Figure 5 3 is a graph showing the signal intensities of collagen fibers and elastic fibers in the dermis of skin explants of each group in Example 3.

[0034] Figure 6 Statistical graphs of collagen fiber signal intensity and elastic fiber signal intensity in the dermis of skin explants in each group in Example 3.

[0035] Figure 7 Statistical graph of heat shock protein HSP70 gene expression indexes in skin explants of each group in Example 4. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0037] The method for evaluating the efficacy of cosmetics in resisting high-temperature heat damage in the present invention comprises the following steps: S1: Disinfect the skin explant in alcohol, clean the blood and adhesions on the skin surface with a balanced salt solution, peel off the subcutaneous tissue, and divide the skin tissue into equal-sized skin slices. Place them on a gelatin sponge pre-soaked with culture medium with the dermis facing down and the epidermis facing up. Add 1.5 ml of DMEM complete culture medium and culture in vitro. S2: The skin explants cultured in vitro were divided into a normal control group, a model control group, and a sample group. After the samples were given and cultured for 24 hours, the model control group and the sample group were subjected to high temperature modeling, and the samples were collected after modeling; S3: The skin explants of each group were cleaned, fixed, embedded, sliced, and then stained to detect the skin morphology and extracellular matrix indicators.

[0038] As a specific embodiment, the skin explant in S1 is washed 2-3 times with a balanced salt solution and 1.5 ml of culture medium is added.

[0039] As a specific embodiment, the culture medium of the sample in S1 is aspirated and discarded, the normal control group and the model control group are replaced with 1.5 mL of fresh culture medium, and the sample group is directly replaced with fresh culture medium containing the sample or replaced with fresh culture medium and 45-50 μL of the sample is dripped or smeared on the surface of the skin explant.

[0040] Preferably, the sample group is administered by liquid phase administration or surface application.

[0041] Furthermore, for samples administered by liquid phase, the medium was replaced with 1.5 mL of fresh culture medium containing a sample of a specified concentration; for samples administered by surface application, the medium was replaced with 1.5 mL of fresh culture medium, and 45-50 μL of a sample of a specified concentration was dripped or applied on the surface of the skin explant.

[0042] As a specific embodiment, in said S2, the step of in vitro culturing is: placing the skin explant in an incubator at 35-37° C. and containing 4-6% CO 2 for culturing.

[0043] Furthermore, the skin explants were placed in an incubator at 37° C. with 5% CO 2 and cultured.

[0044] As a specific embodiment, the model control group and the sample group in S2 are placed in a high-temperature incubator at 55-65°C for 2-4 hours for high-temperature modeling.

[0045] Furthermore, the model control group and sample group were placed in a 65°C high-temperature incubator for 3 h.

[0046] As a specific embodiment, in S3, the skin explant is subjected to one of paraffin embedding and sectioning, OCT embedding and sectioning, skin tissue mRNA extraction and reverse transcription processing.

[0047] As a specific embodiment, in S3, the skin explant is stained with hematoxylin-eosin or Masson's staining.

[0048] As a specific embodiment, in S3, the skin morphology and extracellular matrix indicators are skin tissue morphology, collagen fiber density, collagen fiber and elastic fiber signal intensity, and heat shock protein HSP70 gene expression indicator.

[0049] As a specific embodiment, in S3, the skin morphology and extracellular matrix indicators are photographed using a biological microscope or a two-photon microscope or observed by qPCR.

[0050] Furthermore, skin explants for hematoxylin-eosin staining were fixed, paraffin-embedded, sectioned, stained with hematoxylin-eosin, and observed under a biological microscope. Skin explants for collagen fiber density assays were fixed, paraffin-embedded, sectioned, Masson-stained, and observed under a biological microscope. Skin explants for collagen and elastic fiber expression assays were fixed, dehydrated, OCT-embedded, cryosectioned, and observed under a two-photon microscope. Skin explants for heat shock protein 70 gene expression assays underwent skin tissue mRNA extraction, reverse transcription, and qPCR analysis.

[0051] As a specific embodiment, in S3, Image J software or SPSS26.0 software is used to analyze the skin morphology and extracellular matrix indicators.

[0052] Furthermore, Image J software was used to analyze the collagen fiber density of Masson-stained sections, and Image J software was used to analyze the collagen fiber signal intensity and elastic fiber signal intensity detected by two-photon microscopy. SPSS26.0 software was used for statistical analysis. The statistical analysis results of skin tissue morphology, collagen fiber density, collagen fiber and elastic fiber signal intensity were used to comprehensively evaluate the sample's ability to resist high-temperature thermal damage.

[0053] As a specific embodiment, the expression of the heat shock protein HSP70 gene was detected by extracting mRNA from skin tissue after high-temperature exposure. The expression of the heat shock protein HSP70 gene was detected through reverse transcription and qPCR. The relative expression of the HSP70 gene was statistically analyzed using SPSS 26.0 software.

[0054] The present invention adopts high temperature for modeling, eliminating the possible influence of ultraviolet rays on the evaluation index and evaluation results, and is suitable for the evaluation of the efficacy of cosmetics and their raw materials in resisting high temperature heat damage. Figure 1 shown.

[0055] Example 1 A method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage comprises the following steps: S1: Fresh skin explants were placed in saline and transported quickly and at low temperature to the laboratory for experiments. The skin explants were sterilized in alcohol and then washed three times with a balanced salt solution to remove blood and adhesions from the skin surface. Using surgical scissors and forceps, the subcutaneous tissue was carefully excised and the skin tissue was divided into equal-sized pieces. Absorbable gelatin sponges were cut into equal pieces and placed in a cell culture plate. Culture medium was added and allowed to fully saturate the sponges. The divided skin explants were placed with the dermis facing down and the epidermis facing up on gelatin sponges pre-soaked with culture medium. 1.5 ml of culture medium was added to each well to maintain an air-liquid culture environment for the skin explants. The skin explants were incubated in a 37°C incubator with 5% CO2 until ready for use. Modeling and sample loading were performed the next day. The skin explants were divided into a normal control group, a model control group, and a sample group. The normal and model control groups did not receive sample treatment, while the sample group received sample treatment. Three replicates were performed. The culture medium was aspirated and replaced with 1.5 mL of fresh culture medium for the normal control group and 1.5 mL of fresh culture medium for the model control group. Sample 1 was replaced with 1.5 mL of fresh culture medium containing 0.01% L-ascorbic acid; Sample 2 was replaced with 1.5 mL of fresh culture medium, and 50 μL of 5% recombinant collagen sample was applied to the surface of the skin explant. S2: After culturing the pretreated skin explants for 24 hours, the model control group and sample group were placed in a 65°C high-temperature incubator for 3 hours to establish the model. After the model establishment, the samples were collected; S3: The skin explants of each group were cleaned, fixed, paraffin-embedded, sectioned, and stained with hematoxylin-eosin. The morphological structure of the skin tissues of each group was observed using a biological microscope. Figure 2 shown.

[0056] like Figure 2It can be seen that compared with the normal control group, the stratum corneum of the skin explants in the model control group was severely damaged after high temperature injury, vacuoles appeared between the epidermal cells, the tissue structure was loose, and the number of living epidermal cell layers was reduced, indicating that the high temperature thermal injury model was successful.

[0057] Compared with the model control group, the intercellular vacuoles in the epidermal layer of the skin explants treated with samples 1 and 2 were reduced, the tissue structure and stratum corneum damage were improved, and the number of living epidermal cell layers increased, indicating that samples 1 and 2 have the effect of resisting high temperature thermal damage.

[0058] Example 2 A method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage comprises the following steps: S1: Fresh skin explants were placed in saline and transported quickly and at low temperature to the laboratory for experiments. The skin explants were sterilized in alcohol and then washed three times with a balanced salt solution to remove blood and adhesions from the skin surface. Using surgical scissors and forceps, the subcutaneous tissue was carefully excised and the skin tissue was divided into equal-sized pieces. Absorbable gelatin sponges were cut into equal pieces and placed in a cell culture plate. Culture medium was added and allowed to fully saturate the sponges. The divided skin explants were placed with the dermis facing down and the epidermis facing up on gelatin sponges pre-soaked with culture medium. 1.5 ml of culture medium was added to each well to maintain an air-liquid culture environment for the skin explants. The skin explants were incubated in a 37°C incubator with 5% CO2 until ready for use. Modeling and sample loading were performed the next day. The skin explants were divided into a normal control group, a model control group, and a sample group. The normal and model control groups did not receive sample treatment, while the sample group received sample treatment. Three replicates were performed. The culture medium was aspirated and replaced with 1.5 mL of fresh culture medium for the normal control group and 1.5 mL of fresh culture medium for the model control group. For the sample groups, sample 1 was replaced with 1.5 mL of fresh culture medium containing 0.01% L-ascorbic acid; sample 2 was replaced with 1.5 mL of fresh culture medium, and 50 μL of 5% recombinant collagen sample was applied to the surface of the skin explants. S2: After culturing the pretreated skin explants for 24 hours, the model control group and sample group were placed in a 65°C high-temperature incubator for 3 hours to establish the model. After the model establishment, the samples were collected; S3: The skin explants of each group were cleaned, fixed, paraffin-embedded, sectioned, and Masson-stained. The collagen fiber density of each group was observed using a biological microscope and analyzed using Image J software and SPSS 26.0 software. Figure 3 and Figure 4 shown.

[0059] like Figure 3 and Figure 4As shown in the figure, compared with the normal control group, the collagen fiber density in the dermis of the skin explants in the model control group was significantly reduced after high temperature injury. This means that high temperature disrupts the homeostasis of the extracellular matrix in the dermis, leading to a decrease in collagen fibers. This indicates that the high temperature thermal injury model was successful.

[0060] like Figure 3 and Figure 4 As shown in the results, compared with the model control group, the collagen fiber density in the dermis of the skin explants treated with samples 1 and 2 was significantly increased, indicating that the samples can promote the increase of collagen fibers in the skin damaged by high temperature heat and improve the homeostasis of the dermal extracellular matrix, that is, the samples have the effect of resisting high temperature heat damage.

[0061] Example 3 A method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage comprises the following steps: S1: Fresh skin explants were placed in saline and transported quickly and at low temperature to the laboratory for experiments. The skin explants were sterilized in alcohol and then washed three times with a balanced salt solution to remove blood and adhesions from the skin surface. Using surgical scissors and forceps, the subcutaneous tissue was carefully excised and the skin tissue was divided into equal-sized pieces. Absorbable gelatin sponges were cut into equal pieces and placed in a cell culture plate. Culture medium was added and allowed to fully saturate the sponges. The divided skin explants were placed with the dermis facing down and the epidermis facing up on gelatin sponges pre-soaked with culture medium. 1.5 ml of culture medium was added to each well to maintain an air-liquid culture environment for the skin explants. The skin explants were incubated in a 37°C incubator with 5% CO2 until ready for use. Modeling and sample loading were performed the next day. The skin explants were divided into a normal control group, a model control group, and a sample group. The normal and model control groups did not receive sample treatment, while the sample group received sample treatment. Three replicates were performed. The culture medium was aspirated and replaced with 1.5 mL of fresh culture medium for the normal control group and 1.5 mL of fresh culture medium for the model control group. Sample 1 was replaced with 1.5 mL of fresh culture medium containing 0.01% L-ascorbic acid; Sample 2 was replaced with 1.5 mL of fresh culture medium, and 50 μL of 5% recombinant collagen sample was applied to the surface of the skin explant. S2: After culturing the pretreated skin explants for 24 hours, the model control group and sample group were placed in a 65°C high-temperature incubator for 3 hours to establish the model. After the model establishment, the samples were collected; S3: The skin explants of each group were cleaned, fixed, dehydrated, embedded in OCT, and frozen sectioned. The collagen fiber signal intensity and elastic fiber signal intensity of each group were observed using a two-photon microscope and analyzed using Image J software and SPSS26.0 software. Figure 5 and Figure 6 shown.

[0062] like Figure 5 and Figure 6 As shown in the figure, compared with the normal control group, the signal intensity of collagen fibers and elastic fibers in the dermis of the skin explants in the model control group was significantly reduced after high temperature injury, and the collagen fibers and elastic fibers were broken and sparse. That is, high temperature broke the homeostasis of the extracellular matrix of the skin dermis and affected the assembly of collagen fibers and elastic fibers in the extracellular matrix, indicating that the high temperature thermal injury model was successful.

[0063] like Figure 5 and Figure 6 As shown in the results, compared with the model control group, the signal intensity of collagen fibers and elastic fibers in the dermis of skin explants treated with samples 1 and 2 was significantly increased, indicating that the samples can promote the increase of collagen fibers and elastic fibers in skin damaged by high temperature heat, promote the assembly of collagen fibers and elastic fibers, and maintain the homeostasis of the dermal extracellular matrix, that is, the samples have the effect of resisting high temperature heat damage.

[0064] Example 4 A method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage comprises the following steps: S1: Fresh skin explants were placed in saline and transported quickly and at low temperature to the laboratory for experiments. The skin explants were sterilized in alcohol and then washed three times with a balanced salt solution to remove blood and adhesions from the skin surface. Using surgical scissors and forceps, the subcutaneous tissue was carefully excised and the skin tissue was divided into equal-sized pieces. Absorbable gelatin sponges were cut into equal pieces and placed in a cell culture plate. Culture medium was added and allowed to fully saturate the sponges. The divided skin explants were placed with the dermis facing down and the epidermis facing up on gelatin sponges pre-soaked with culture medium. 1.5 ml of culture medium was added to each well to maintain an air-liquid culture environment for the skin explants. The skin explants were incubated in a 37°C incubator with 5% CO2 until ready for use. Modeling and sample loading were performed the next day. The skin explants were divided into a normal control group, a model control group, and a sample group. The normal and model control groups did not receive sample treatment, while the sample group received sample treatment. Three replicates were performed. The culture medium was aspirated and replaced with 1.5 mL of fresh culture medium for the normal control group and 1.5 mL of fresh culture medium for the model control group. For the sample groups, sample 1 was replaced with 1.5 mL of fresh culture medium containing 0.01% L-ascorbic acid; sample 2 was replaced with 1.5 mL of fresh culture medium, and 50 μL of 5% recombinant collagen sample was applied to the surface of the skin explants. S2: After culturing the pretreated skin explants for 24 hours, the model control group and sample group were placed in a 65°C high-temperature incubator for 3 hours to establish the model. After the model establishment, the samples were collected; S3: Extract skin tissue mRNA, and detect the expression of heat shock protein HSP70 gene through reverse transcription and qPCR. SPSS26.0 software was used to perform statistical analysis on the relative expression of HSP70 gene. Figure 7 shown.

[0065] like Figure 7 As shown in the figure, compared with the normal control group, the relative expression of the heat shock protein HSP70 gene in the skin explants of the model control group increased significantly after high temperature injury, indicating that the high temperature heat injury model was successfully established. Compared with the model control group, the relative expression of the heat shock protein HSP70 gene in the skin explants treated with sample 1 was significantly reduced, indicating that the sample can inhibit the expression of the heat shock protein HSP70, that is, the sample has the effect of resisting high temperature heat injury.

[0066] Compared with the normal control group, the relative expression of the heat shock protein HSP70 gene in the skin explants of the model control group significantly increased after high-temperature injury, indicating that the high-temperature thermal injury model was successfully established. Compared with the model control group, the relative expression of the heat shock protein HSP70 gene in the skin explants treated with Sample 2 was significantly reduced, indicating that the sample can inhibit the expression of the heat shock protein HSP70, that is, the sample has the effect of resisting high-temperature thermal injury.

[0067] The experimental model of the present invention is a "skin explant", which refers to donated or discarded human skin tissue obtained from clinical practice and cultivated in a specific way to form an efficacy test model. The "skin explant" is completely consistent with the real in vivo skin structure, including the stratum corneum, epidermis, DEJ layer, dermis, immune cells, etc., while the structure and function of the 3D epidermal model + fibroblast co-culture model are significantly different from those of real human skin tissue (epidermis + dermis), such as the lack of DEJ layer wrinkles, the lack of dense tissue structure of the dermis and the extracellular matrix. Therefore, the present invention can directly observe the expression of collagen fibers and elastic fibers in skin explants (signal strength) through two-photon microscopy, while the models in existing patents cannot achieve this effect.

[0068] In summary, the method for evaluating the efficacy of cosmetics and their raw materials in resisting high-temperature thermal damage of the present invention can effectively evaluate the efficacy of cosmetics and their raw materials in resisting high-temperature thermal damage by analyzing skin tissue morphology, collagen fiber density, collagen fiber and elastic fiber signal intensity, and heat shock protein HSP70 gene expression evaluation index. The experimental results are visualized and easy to understand. The hematoxylin-eosin staining method can directly observe the morphological structure of the dermis and epidermis of the skin explant and the damaging effect of high temperature on the skin. The Masson staining method can directly observe the density of collagen fibers in the skin explant. The two-photon microscopy imaging method can directly observe the expression (signal strength) of collagen fibers and elastic fibers in the skin explant. The method has high authenticity and strong intuitiveness, can directly reflect the efficacy of the product or raw material in resisting high-temperature thermal damage on the skin, and has a wide range of applicability, not only applicable to finished cosmetics and raw materials, but also applicable to samples with poor solubility such as oil solubility.

[0069] Those skilled in the art should recognize that the above embodiments are merely intended to illustrate the present invention and are not intended to limit the present invention. As long as they are within the spirit of the present invention, any changes or modifications to the above embodiments will fall within the scope of the claims of the present invention.

Claims

1. A method for evaluating the efficacy of cosmetics in resisting high-temperature heat damage, characterized in that: The following steps are involved: S1: After disinfecting the skin explant, clean the blood and adhesions on the skin surface with a balanced salt solution, peel off the subcutaneous tissue, and divide the skin tissue into equal-sized skin slices. Place them with the dermis facing down and the epidermis facing up on a gelatin sponge pre-soaked with culture medium, add culture medium, and culture in vitro; S2: The skin explants cultured in vitro were divided into a normal control group, a model control group, and a sample group. After the samples were given and cultured for 24 hours, the model control group and the sample group were subjected to high temperature modeling, and the samples were collected after modeling; S3: The skin explants of each group were cleaned, fixed, embedded, sliced, and then stained to detect the skin morphology and extracellular matrix indicators.

2. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 1, characterized in that: The skin explants in S1 are washed 2-3 times with a balanced salt solution, and an appropriate amount of culture medium is added.

3. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 1, characterized in that: The culture medium of the samples in S1 was aspirated and discarded, and the normal control group and the model control group were replaced with 1.5 mL of fresh culture medium. The sample group was directly replaced with fresh culture medium containing the sample or replaced with fresh culture medium and 45-50 μL of sample was dripped or smeared on the surface of the skin explant.

4. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 3, characterized in that: The concentration of the sample is 0.01%-5% or 0.1%-5%.

5. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 1, characterized in that: In said S2, the step of in vitro culturing is: placing the skin explant in an incubator at 35-37° C. and containing 4-6% CO 2 for culturing.

6. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 1 or 4, characterized in that: In S2, the model control group and the sample group were placed in a 55-65° C. high-temperature incubator for 2-4 hours of high-temperature modeling.

7. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 1, characterized in that: In S3, the skin explant is subjected to paraffin embedding and sectioning or OCT embedding and sectioning, or skin tissue mRNA is extracted and reverse transcribed.

8. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 1, characterized in that: In S3, skin explants were stained with hematoxylin-eosin or Masson's staining.

9. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 1, characterized in that: In said S3, said skin morphology and extracellular matrix indicators are skin tissue morphology, collagen fiber density, collagen fiber and elastic fiber signal intensity, and heat shock protein HSP70 gene expression indicator.

10. The method for evaluating the efficacy of cosmetics in resisting high-temperature thermal damage according to claim 8, characterized in that: In S3, the skin morphology and extracellular matrix indicators are photographed using a biological microscope or a two-photon microscope or observed by qPCR.

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