Neurodermatitis model constructed by utilizing capsaicin stimulation as well as construction method and application of neurodermatitis model

Abstract

The invention discloses a neurodermatitis model constructed by capsaicin stimulation and a construction method and application thereof, and belongs to the technical field of cosmetic raw material function testing. According to the application, the 3D skin model is stimulated by using capsaicin, and the culture solution of the 3D skin model is co-cultured with mast cells and vascular endothelial cells, so that the neurodermatitis model capable of simulating nerve-immunity-skin barrier-blood vessel complex interaction is successfully constructed in vitro. Based on the model, a multi-index joint detection and weighted comprehensive scoring system covering four dimensions of nerves, inflammations, skin barriers and vascular reactions is also established, and deep, systematic and quantitative evaluation of the in-vitro neurodermatitis relieving and repairing effects of cosmetic raw materials is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of functional test of cosmetic raw materials, and particularly relates to a neurodermatitis model constructed by stimulating capsaicin and a construction method and application thereof. BACKGROUND

[0002] In the development process of efficacy cosmetics for sensitive skin, it is crucial to establish a precise and reliable soothing and repairing efficacy evaluation system. The evaluation system not only serves the raw material screening and performance prediction in the formula development stage, but also is the core scientific basis for supporting the anti-sensitivity, redness reduction, and barrier repair efficacy claims of products before they are put on the market. Therefore, developing an in vitro model that can truly simulate the complex pathological state of sensitive skin and achieve multi-dimensional precise evaluation has become an urgent need for the industry development.

[0003] At present, the commonly used evaluation model has significant shortcomings in simulating sensitive skin, which restricts the development efficiency and scientificity of related products.

[0004] Animal experiments face severe ethical challenges and social controversies, and their application is strictly limited. In addition, there are essential differences between animals and humans in terms of skin physiological structure and stress response mechanism, which limits the predictive value of experimental results to the human body. Animal experiments are also characterized by high cost and long cycle, which makes it difficult to meet the needs of efficient research and development.

[0005] Traditional two-dimensional cell models cannot simulate the three-dimensional structure and barrier function of human skin, and have fundamental limitations. Laboratory studies have shown that even if HaCaT keratinocytes or TRPV1 overexpressed cell lines are used, due to the lack of a three-dimensional growth environment, cells cannot differentiate directionally, and thus cannot induce the release of P substance, a key mediator of neurogenic inflammation. This defect makes 2D models difficult to effectively simulate the core neuro-immune co-regulation mechanism of sensitive skin, leading to a serious distortion of the efficacy evaluation of soothing raw materials.

[0006] In sharp contrast to 2D models, 3D skin models, with their unique three-dimensional structure, including the complete structure of the epidermis-korium, granular layer, stratum spinosum, and basal layer, provide a growth microenvironment closer to the in vivo environment for cells, support and induce directional differentiation of cells, and keratinocytes with differentiation ability have the ability to release P substance, making it possible to construct a neurogenic inflammation model.

[0007] Although three-dimensional skin models can better simulate skin structure, existing technical solutions have two major shortcomings: Existing technologies generally use chemical stimulants such as sodium dodecyl sulfate, which mainly induce damage by disrupting the chemical barrier, and cannot comprehensively simulate the complex pathological state involving abnormal conduction of nerve signals, immune inflammatory response, and other multi-pathway interweaving in sensitive skin.

[0008] Evaluation index is one-sided: existing efficacy evaluation is mostly limited to detecting individual inflammatory factors, and the index is single, which cannot cover the multi-dimensional phenotypes of sensitive skin such as itching, stinging, burning, redness and barrier damage. Lack of systematic evaluation system leads to one-sided data, making it difficult to accurately and reliably evaluate the comprehensive soothing and repairing efficacy of cosmetics.

[0009] In addition, existing commercial skin models on the market, such as EpiDerm, SkinEthic, EpiSkin, etc., although they simulate some structures and functions of the skin to some extent, they generally lack standardized sensitive skin modeling schemes. This makes it difficult to compare and consistent the experimental results due to differences in modeling methods when different laboratories use these models for sensitive skin research, which seriously hinders the in-depth development and achievement transformation of related research.

[0010] Therefore, it is necessary to construct a new sensitive damaged neurodermatitis model in vitro efficacy verification method, and develop a multi-dimensional evaluation and assessment system, so as to more efficiently and accurately realize the soothing and repairing efficacy development and screening of cosmetics. SUMMARY

[0011] In order to solve the above technical problems, the present application provides a neurodermatitis model constructed by stimulating capsaicin and its construction method and application.

[0012] In a first aspect, the present application provides a neurodermatitis model constructed by stimulating capsaicin, which comprises: a 3D skin model stimulated and incubated by capsaicin; and a culture solution of the 3D skin model after being stimulated and incubated by capsaicin; a mast cell co-cultured with the culture solution of the 3D skin model after being stimulated and incubated by capsaicin and a culture solution of the co-cultured mast cell; a vascular endothelial cell co-cultured with the culture solution of the 3D skin model after being stimulated and incubated by capsaicin and a culture solution of the co-cultured vascular endothelial cell.

[0013] Further, in some embodiments, the 3D skin model comprises: a human recombinant 3D epidermis model or a human recombinant 3D full skin model; preferably, the 3D skin model comprises: EpiSkin TM , SkinEthic TM RHE, T-Skin TM 3D skin model, EpiDerm TM 3D skin model.

[0014] Further, in some embodiments, the model is used to evaluate neurodermatitis; neurodermatitis has one or more of the following characteristic dimensions A-D detectable: Characteristic dimension A: neurogenic inflammation response characteristic dimension; Characteristic dimension B: immune response and inflammation response characteristic dimension; Characteristic dimension C: skin barrier function characteristic dimension; Characteristic dimension D: vascular response characteristic dimension; Wherein, the 3D skin model stimulated by capsaicin and incubated is used to reflect one or more of the following characteristic dimensions A, B, C, D of neurodermatitis: The culture solution of the 3D skin model after being stimulated by capsaicin and incubated is used to reflect one or more of the following characteristic dimensions A, B, D of neurodermatitis: The mast cell culture solution after being co-cultured with the culture solution of the 3D skin model after being stimulated by capsaicin and incubated is used to reflect the characteristic dimension D of neurodermatitis; The vascular endothelial cells co-cultured with the culture solution of the 3D skin model after being stimulated by capsaicin and incubated and the culture solution of the co-cultured vascular endothelial cells are used to reflect the characteristic dimension D of neurodermatitis.

[0015] Further, in some embodiments, the characteristic dimension A is embodied by one or more of the following indicators: The expression amount of nerve sensitivity and perception related genes; the nerve sensitivity and perception related genes include one or more of TRPV1, TRPV2, TRPA1, ASIC3, SCN9A; The expression amount of neuropeptide and neuropeptide related receptor genes; the neuropeptides include one or more of substance P, CGRP, VIP, NPY; the neuropeptide related receptor genes include one or more of substance P receptor NK-1R, CGRPR; The secretion amount of neuropeptide related factors; the neuropeptide related factors include one or more of substance P, CGRP, VIP, NPY factors; The expression amount of keratin formation related genes; the keratin formation related genes include one or more of KRT1, KRT10, KRT14, KRT16, KRT17, KLK5; The expression amount of TRP receptor family proteins; the TRP receptor family proteins include one or more of TRPV1, TRPA1; The expression amount of neuropeptide related receptor proteins; the neuropeptide related receptor proteins include one or more of NK-1R, CGRPR proteins.

[0016] Further, in some embodiments, the characteristic dimension A is embodied by one or more of the following indicators:

[0017] Further, in some embodiments, the characteristic dimension B is embodied by one or more of the following indicators: expression level of an inflammation response related gene; the inflammation response related gene includes one or more of IL-1a, IL-1b, IL-6, IL-10, IL-12b, IL-12, IL-17, IL-22, IL-33, TNF-a, CXCL8; expression level of an immune regulation related gene; the immune regulation related gene includes one or more of TLR2, TLR4, TLR9, NFKB1, NFKB2; secretion level of a skin swelling and inflammation related factor; the skin swelling and inflammation related factor includes one or more of IL-1a, IL-1b, IL-6, CXCL8, IL-12, IL-17, IL-22, IL-33, TNF-a factor.

[0018] Further, in some embodiments, the characteristic dimension B is embodied by one or more of the following indicators:

[0019] Further, in some embodiments, the characteristic dimension C is embodied by one or more of the following indicators: expression level of a skin barrier structure protein gene; the skin barrier structure protein gene includes one or more of FLG, LOR, IVL, TGM1, CLDN1, OCLN, DSP, DSG, ZO-1, SPINK5; expression level of a dry desquamation related protease and inhibitor gene; the dry desquamation related protease and / or inhibitor gene includes one or more of KLK7, MMP9, MMP1; skin physiological function indicator; the skin physiological function indicator includes one or more of transepidermal water loss value, epidermal thickness; expression level of a skin barrier structure protein; the skin barrier structure protein includes one or more of FLG, LOR, IVL, DSG protein; expression level of a tight junction protein; the tight junction protein includes one or more of CLDN1, ZO-1 protein.

[0020] Further, in some embodiments, the characteristic dimension C is embodied by one or more of the following indicators: the expression level of skin barrier structure protein genes, the expression level of skin barrier structure proteins, the down-regulation of the expression level of tight junction proteins; the up-regulation of the expression level of dry desquamation related protease and / or inhibitor genes; the deterioration of skin physiological function indicators, including a significant increase in trans-epidermal water loss value and a significant decrease in epidermal thickness.

[0021] Further, in some embodiments, the characteristic dimension D is embodied by one or more of the following indicators: The amount of histamine released in the mast cell culture solution after co-culture; The expression level of blood vessel response related genes; the blood vessel response related genes include one or more of the following: ETBR, VEGFR2, TIE2, PECAM-1, ICAM-1, VCAM-1, NOS3, and ET-1 genes; The secretion amount of blood vessel response related factors; the blood vessel response related factors include one or more of the following: NO, ET-1, ICAM-1, and VCAM-1 factors; The secretion amount of blood vessel response related growth factors; the blood vessel response related growth factors include one or more of the following: VEGF, EGF, FGF, and PDGF factors.

[0022] Further, in some embodiments, the characteristic dimension D is embodied by one or more of the following indicators: the up-regulation of the amount of histamine released, the secretion amount of blood vessel response related factors, and the secretion amount of blood vessel response related growth factors; and the down-regulation of the expression level of blood vessel response related genes.

[0023] Further, in some embodiments, the indicators in the characteristic dimensions A, B, C, and D are selected, and the total number of selected indicators is more than 3.

[0024] In a second aspect, the present application provides a method for constructing a neurodermatitis model stimulated by capsaicin, which comprises the following steps: stimulating a 3D skin model with capsaicin, incubating the stimulated 3D skin model, co-culturing the culture solution of the incubated 3D skin model with mast cells and vascular endothelial cells, and obtaining the neurodermatitis model.

[0025] Further, in some embodiments, the method for constructing a neurodermatitis model stimulated by capsaicin comprises the following steps: Stimulating a 3D skin model with a capsaicin working solution, incubating the stimulated 3D skin model in a culture solution, and collecting the incubated 3D skin model and the culture solution of the 3D skin model; The culture solution of the incubated 3D skin model is prepared into mast cell conditioned medium with mast cell culture medium, the mast cells are cultured using the mast cell conditioned medium, and the cultured mast cells and mast cell culture solution are collected; The culture solution of the incubated 3D skin model is prepared into vascular endothelial cell conditioned medium with vascular endothelial cell culture medium, the vascular endothelial cells are cultured using the vascular endothelial cell conditioned medium, and the cultured vascular endothelial cells and vascular endothelial cell culture solution are collected.

[0026] Further, in some embodiments, the working concentration of capsaicin is 0.1-3 mM.

[0027] Further, in some embodiments, EpiSkin TM The working concentration of capsaicin in the 3D skin model is 2-3 mM; SkinEthic TM The working concentration of capsaicin in the RHE 3D skin model is 0.1-0.3 mM; T-Skin TM The working concentration of capsaicin in the 3D skin model is 3-5 mM; EpiDerm TM The working concentration of capsaicin in the 3D skin model is 1-2 mM.

[0028] Further, in some embodiments, the incubation conditions of the stimulated 3D skin model, the culture conditions of the mast cells and the vascular endothelial cells are all 37±5℃, (5±1)% CO2, and saturated humidity.

[0029] Further, in some embodiments, the incubation time of the stimulated 3D skin model is 12-48 hours; and the culture time of the mast cells and the vascular endothelial cells is 24-36 hours.

[0030] Further, in some embodiments, the volume ratio of the culture solution of the incubated 3D skin model to the mast cell culture medium or the vascular endothelial cell culture medium is 1: (1-3).

[0031] In a third aspect, the present application provides a method for evaluating the in-vitro neurodermatitis soothing and repairing effect of a cosmetic raw material, which uses the neurodermatitis model constructed by stimulating with capsaicin according to the first aspect, and is characterized by comprising the following steps: S1: Take 3D skin models, and set up a blank control group, a negative control group, a positive control group, and a sample group; except for the blank control group, the negative control group, the positive control group, and the sample group are respectively stimulated and induced using capsaicin working solution to treat the 3D skin models; and the 3D skin models in each group after the stimulation and induction are placed in culture solution; S2: The blank control group set in step S1 is not treated with drug; fresh culture medium is added in the negative control group; positive drug is added in the positive control group; the sample group is added with the sample to be tested; then the 3D skin models in each group are respectively incubated; the 3D skin models and the culture solution of each group after incubation are collected respectively; S3: The expression amount of different genes in the 3D skin model cells after incubation, the secretion amount of different factors in the culture solution of the 3D skin model after incubation, the skin thickness, trans-epidermal water loss and protein expression amount of the 3D skin model after incubation are detected; the relative expression amount of different genes in the 3D skin model cells of each group is calculated respectively; the relative secretion amount of different factors in the culture solution of the 3D skin model of each group is calculated respectively; the skin thickness, trans-epidermal water loss and protein relative expression amount of the 3D skin model of each group are calculated respectively; S4: The mast cells are cultured with the conditioned medium; the culture solution of each group of 3D skin models after incubation collected in step S2 is mixed with the mast cell culture medium to prepare the mast cell conditioned medium; the mast cells are cultured with the mast cell conditioned medium; the mast cells after culture and the culture solution of the mast cells are collected respectively; S5: The histamine release amount in the culture solution of each group of mast cells after culture in step S4 is detected; the relative release amount of histamine in the culture solution of each group of mast cells after culture is calculated; S6: The vascular endothelial cells are cultured with the conditioned medium; the culture solution of each group of 3D skin models after incubation collected in step S2 is mixed with the vascular endothelial cell culture medium to prepare the vascular endothelial cell conditioned medium; the vascular endothelial cells are cultured with the vascular endothelial cell conditioned medium; the vascular endothelial cells after culture and the culture solution of the vascular endothelial cells are collected respectively; S7: The expression amount of different genes in the vascular endothelial cells after culture in step S6, the secretion amount of different factors in the culture solution of the vascular endothelial cells after culture are detected; the relative expression amount of different genes in the vascular endothelial cells after culture of each group is calculated respectively; the relative secretion amount of different factors in the culture solution of the vascular endothelial cells after culture of each group is calculated respectively; S8: The relative expression amount of different genes in the 3D skin model cells after incubation, the relative secretion amount of different factors in the culture solution of the 3D skin model after incubation, the protein relative expression amount, the skin thickness and the trans-epidermal water loss in step S3 are statistically analyzed respectively; The relative release amount of histamine in the culture solution of each group of mast cells after culture in step S5 is statistically analyzed respectively; The relative expression amount of different genes and the relative secretion amount of different factors in step S7 are statistically analyzed respectively; According to the differences of the corresponding detection indexes of the sample group and the negative control group, the detection indexes of the sample group are scored respectively; S9: The scores of the detection indexes of the sample group are comprehensively evaluated to evaluate the in-vitro neurodermatitis soothing and repairing effect of the cosmetic raw material.

[0032] Further, in some embodiments, according to the differences of the corresponding detection indexes of the sample group and the negative control group, the detection indexes of the sample group are scored respectively, and the scoring standard is that the p value between the corresponding detection indexes of the sample group and the negative control group is less than 0.05, a slight significant difference, 0.3 points; p<0.01, a significant difference, 0.5 points; p<0.001, a very significant difference, 0.7 points; P<0.0001, an extremely significant difference, 1.0 points.

[0033] Further, in some embodiments, the scores of the detection indexes of the sample group are comprehensively evaluated to evaluate the in-vitro neurodermatitis soothing and repairing effect of the cosmetic raw material, which is obtained by weighted comprehensive evaluation of the scores of the indexes in the sample group according to the weight distribution of characteristic dimensions A to D. The evaluation of the in-vitro neurodermatitis soothing and repairing effect of the cosmetic raw material is obtained by weighted comprehensive evaluation of the scores of the indexes in the sample group according to the weight distribution of characteristic dimensions A to D. The weight distribution of characteristic dimensions A to D is as follows: Characteristic dimension A: the weight coefficient of the neurogenic inflammation characteristic dimension is 30%; Characteristic dimension B: the weight coefficient of the immune response and inflammation reaction characteristic dimension is 30%; Characteristic dimension C: the weight coefficient of the skin barrier function characteristic dimension is 25%; Characteristic dimension D: the weight coefficient of the vascular response characteristic dimension is 15%.

[0034] Further, in some embodiments, the blank control group, the negative control group, the positive control group and the sample group are respectively provided with 3 parallel groups; the sample group is at least one sample group; and the sample group can be provided with multiple sample groups.

[0035] Further, in some embodiments, the working solution concentration of capsaicin is 0.1-3 mM, and the addition amount is 1-2 ml per well.

[0036] Further, in some embodiments, EpiSkin TM The working solution concentration of capsaicin in the 3D skin model is 2-3 mM; SkinEthic TM The working solution concentration of capsaicin in the RHE 3D skin model is 0.1-0.3 mM; T-Skin TM The working solution concentration of capsaicin in the 3D skin model is 3-5 mM; EpiDerm TM The working solution concentration of capsaicin in the 3D skin model is 1-2 mM.

[0037] Further, in some embodiments, the positive drug is capsaicin.

[0038] Further, in some embodiments, the working solution concentration of the positive drug is 40-1000 μM, and the added amount is 1-2 ml per well; wherein, EpiSkin TM The working solution concentration of the positive drug in the 3D skin model is 400-600 μM; SkinEthic TM The working solution concentration of the positive drug in the RHE 3D skin model is 40-60 μM; T-Skin TM The working solution concentration of the positive drug in the 3D skin model is 600-1000 μM; EpiDerm TM The working solution concentration of the positive drug in the 3D skin model is 100-200 μM.

[0039] Further, in some embodiments, the incubation conditions of the 3D skin models in each group in step S2 are temperature 37±5℃, (5±1)% CO2, saturated humidity, and the culture time is 12-48 hours.

[0040] Further, in some embodiments, the culture solution of the incubated 3D skin models in each group is prepared into mast cell conditioned medium with mast cell culture medium, wherein the volume ratio of the culture solution of the incubated 3D skin models in each group to the mast cell culture medium is 1:(1-3).

[0041] Further, in some embodiments, the culture solution of the incubated 3D skin models in each group is prepared into vascular endothelial conditioned medium with vascular endothelial cell culture medium, wherein the volume ratio of the culture solution of the incubated 3D skin models in each group to the vascular endothelial cell culture medium is 1:(1-3).

[0042] Further, in some embodiments, the incubation conditions of the mast cells or the vascular endothelial cells are temperature 37±5℃, (5±1)% CO2, saturated humidity; and the incubation time is 24-36 hours.

[0043] Further, in some embodiments, the detection of the gene expression amount in the culture solution of the incubated 3D skin models in each group is to detect the gene content in each group of culture solutions by using an ELISA kit.

[0044] Further, in some embodiments, the detection of the skin thickness of the incubated 3D skin models in each group is to perform staining on the skin slices by using the H&E staining method, take photos under a microscope, and measure the thickness of the skin.

[0045] Further, in some embodiments, the detection of the skin thickness of the incubated 3D skin models in each group is to perform 7-10 times of random measurement on each skin slice at different fields and different positions.

[0046] Further, in some embodiments, the detection of the skin thickness of each group of 3D skin models after incubation is specifically operated as follows: the skin tissue is fixed and gradient dehydrated, the skin tissue is embedded with paraffin, the glass slide is stained with hematoxylin and eosin after slicing, the glass slide is sealed with resin after staining, and the glass slide is photographed under a microscope.

[0047] Further, in some embodiments, the detection of the transdermal water loss of each group of 3D skin models after incubation is to measure the transdermal water loss value of the skin using a transdermal water loss measuring instrument (ASCH).

[0048] Further, in some embodiments, the detection of the transdermal water loss of each group of 3D skin models after incubation is to measure the transdermal water loss value of the skin using a transdermal water loss measuring instrument (ASCH).

[0049] Further, in some embodiments, the detection of the transdermal water loss of each group of 3D skin models after incubation is specifically operated as follows: the skin is placed at room temperature for a certain period of time, the instrument is pressed on the top of the skin culture cup to form a closed space, and the transdermal water loss value is measured.

[0050] Further, in some embodiments, the detection of the protein expression amount of each group of 3D skin models after incubation is to use an immunofluorescence staining method.

[0051] Further, in some embodiments, the detection of the protein expression amount of each group of 3D skin models after incubation is specifically operated as follows: the skin tissue is fixed and gradient dehydrated, the skin tissue is embedded with paraffin, the glass slide is sealed, the first antibody is dyed, the second antibody is dyed, and the nucleus is dyed after slicing, the glass slide is sealed with an anti-fluorescence quenching sealing agent after staining, and the glass slide is photographed using a fluorescence microscope.

[0052] Further, in some embodiments, the detection of the protein expression amount of each group of 3D skin models after incubation is to randomly photograph 7-10 times at different fields of view and different positions of each skin slice; the fluorescence intensity of the picture is counted using Image J analysis software, and the relative fluorescence intensity of each group is calculated based on the standard of the negative control group.

[0053] Further, in some embodiments, the detection of the amount of histamine released in the culture solution of each group of mast cells after culture is to detect the amount of histamine released in the culture solution of each group of mast cells using an ELISA kit.

[0054] Furthermore, in some embodiments, the expression levels of different genes related to vascular response in vascular endothelial cells of each group after culture were detected by extracting RNA from vascular endothelial cells of each group using the Trizol method, performing reverse transcription on the extracted RNA using a kit, and then detecting the gene expression levels using real-time quantitative PCR.

[0055] Furthermore, in some embodiments, the secretion levels of different vascular response factors in the culture medium of each group of vascular endothelial cells after culture are detected by using an ELISA kit.

[0056] Furthermore, in some embodiments, 2^ The △△Ct method was used to calculate the relative expression levels of different genes in the 3D skin model cells of each group after incubation, and the expression levels of different genes for vascular response in the vascular endothelial cells of each group.

[0057] Furthermore, in some embodiments, using the negative control group as a benchmark, the relative secretion of different factors in the culture medium of each group of 3D skin models after incubation, the relative release of histamine in the culture medium of each group of mast cells after culture, and the relative secretion of different vascular response factors in the culture medium of each group of vascular endothelial cells after culture were calculated.

[0058] Furthermore, in some embodiments, the statistical analysis employs one-way ANOVA statistical analysis.

[0059] Furthermore, in some embodiments, the different genes in the 3D skin model cells after incubation include one or more of the following: genes related to nerve sensitivity and perception: TRPV1, TRPV2, TRPA1, ASIC3, SCN9A; genes related to dryness and desquamation: KLK5, KLK7, MMP9; genes related to inflammation response: IL-1α, IL-1β, IL-6, IL-10, IL-12β, IL-12, IL-17, IL-22, IL-33, TNF-α, CXCL8; and skin barrier structural proteins. One or more of the following genes: FLG, LOR, IVL, TGM1, CLDN1, OCLN, DSP, DSG, ZO-1, SPINK5; one or more of the following keratinization-related genes: KRT1, KRT10, KRT14, KRT16, KRT17, KLK5; one or more of the following immune regulation-related genes: TLR2, TLR4, TLR9, NFKB1, NFKB2; one or more of the following neuropeptides: substance P, CGRP, VIP, NPY; one or more of the following neuropeptide-related receptor genes: substance P receptor NK-1R, CGRPR.

[0060] Further, in some embodiments, the different factors in the culture solution of each group of 3D skin models after incubation include one or more of the following: neuropeptide substance P, CGRP, VIP, NPY; skin swelling and inflammation-related factors IL-1α, IL-1β, IL-6, CXCL8, IL-12, IL-17, IL-22, IL-33, TNF-α; blood vessel response-related growth factors VEGF, EGF, FGF, PDGF; neuropeptide-related factors substance P, CGRP, VIP, NPY. Further, in some embodiments, the proteins of each group of 3D skin models after incubation include one or more of the following: skin barrier structure protein genes FLG, LOR, IVL, DSG; TRP receptor family proteins including one or more of TRPV1, TRPA1; tight junction proteins CLDN1, ZO-1; neuropeptide-related receptor proteins including one or more of substance P receptor protein NK-1R, CGRPR protein.

[0061] Further, in some embodiments, the different blood vessel response-related genes in each group of cultured vascular endothelial cells include one or more of the following: ETBR, VEGFR2, TIE2, PECAM-1, ICAM-1, VCAM-1, NOS3, ET-1.

[0062] Further, in some embodiments, the different blood vessel response-related factors in the culture solution of each group of cultured vascular endothelial cells include one or more of the following: VEGF, NO, ET-1, ICAM-1, VCAM-1.

[0063] It should be noted that the order of steps S4 and S6 of the third aspect can be changed, that is, the operation of culturing mast cells in conditioned medium can be performed first, or the operation of culturing vascular endothelial cells in conditioned medium can be performed first. When the order of steps S4 and S6 is changed, the corresponding steps S5 and S7 are also adjusted accordingly.

[0064] Further, in some embodiments, the method for evaluating the in-vitro neurodermatitis soothing and repairing effect of the cosmetic raw material further comprises the following steps before step S1: S0: 3D skin model, mast cell, vascular endothelial cell, and pretreatment of the sample to be tested, including the following steps: S0-1: Place the 3D skin model in a culture plate, add culture solution, and culture; S0-2: Seed the mast cells into the culture plate and culture until the mast cells adhere and stably proliferate; S0-3: Seed the vascular endothelial cells into the culture plate and culture until the vascular endothelial cells adhere and stably proliferate. S0-4: Dissolve the sample to be tested in the culture medium or organic solvent to prepare a sample solution to be tested.

[0065] The sample to be tested includes a positive drug and a sample of a raw material to be tested.

[0066] Further, in some embodiments, in step S0-1, the amount of culture medium added is 1-2 ml per well; the culture conditions are 37±5℃, (5±1)% CO2, and saturated humidity; and the culture time is 10-14 hours.

[0067] Step S0-1 is used to stabilize the culture of the skin before the experiment. In step S1, fresh culture medium is replaced to carry out the capsaicin stimulation-induced 3D skin model experiment.

[0068] Further, in some embodiments, in step S0-2, the amount of mast cells seeded into the culture plate is 1*10^6-5*10^6 cells per well; the culture conditions are 37±5℃, (5±1)% CO2, and saturated humidity.

[0069] Further, in some embodiments, in step S0-2, the culture until the mast cells adhere and stably proliferate means that the culture is until the mast cells adhere and grow to about 90% confluence.

[0070] Further, in some embodiments, in step S0-3, the amount of vascular endothelial cells seeded into the culture plate is 5*10^5-5*10^6 cells per well; the culture conditions are 37±5℃, (5±1)% CO2, and saturated humidity.

[0071] Further, in some embodiments, in step S0-3, the culture until the vascular endothelial cells adhere and stably proliferate means that the culture is until the vascular endothelial cells adhere and grow to about 90% confluence.

[0072] Further, in some embodiments, in step S0-4, when the solubility of the sample to be tested in water is above 1000 μg / mL, the sample to be tested is directly dissolved in the culture medium; when the solubility of the sample to be tested in water is below 1000 μg / mL, the sample to be tested is dissolved in an organic solvent, and the volume of the organic solvent should not exceed 1% of the total volume of the culture medium; when an organic solvent is used to dissolve the sample of the raw material to be tested, the same proportion of organic solvent needs to be added to the culture medium of the blank control group without capsaicin stimulation induction to ensure consistency of the matrix.

[0073] In a fourth aspect, the present application provides a screening method of cosmetic raw materials with soothing and repairing efficacy for neurodermatitis, which uses the in vitro evaluation method of soothing and repairing efficacy for neurodermatitis of cosmetic raw materials in the third aspect to evaluate the soothing and repairing efficacy for neurodermatitis of the raw material sample to be tested, and selects the raw material showing the soothing and repairing efficacy for neurodermatitis.

[0074] Compared with the prior art, the technical scheme of the present application has the following beneficial technical effects: The pathological mechanism simulation is more accurate and more comprehensive: the combination of capsaicin stimulation and 3D skin model successfully simulates the complex network of multiple interactions of the core pathological links of neurodermatitis of the nerve-immune-skin barrier-vascular in vitro. The model not only reproduces the skin barrier damage, but more importantly, triggers neurogenic inflammation and related vascular response, making it highly consistent with the mechanism of sensitive skin in phenotype.

[0075] The technical platform has strong universality and high standardization: the model construction method established is suitable for at least four mainstream commercial 3D skin models such as EpiSkin™, EpiDerm™, breaking through the limitation of the prior art which is usually only applicable to a single specific model. This multi-model compatibility ensures the repeatability and stability of the model construction method under different laboratory conditions, while effectively shortening the detection period and reducing the research and development cost.

[0076] The evaluation system has a deeper dimension and more reliable data: the present application establishes a joint detection standard covering four core dimensions of neurogenic inflammation, immune response, skin barrier function and vascular response, which can comprehensively evaluate the action target and efficacy of the test substance on different sensitive pathways, greatly improving the depth and breadth of evaluation. Further, a multi-index comprehensive scoring system based on weight coefficients is introduced to convert complex multi-dimensional data into quantifiable efficacy strength values. The evaluation method of in vitro soothing and repairing efficacy for neurodermatitis of cosmetic raw materials provided by the present application is verified using positive drugs with known efficacy, and the results show that it can accurately and sensitively reflect the efficacy, significantly enhancing the scientificity and reliability of the evaluation results.

[0077] The application prospect is clear and the conversion value is high: the present application provides a systematic and quantitative solution to the soothing and repairing efficacy evaluation of raw materials in the field of cosmetics, thereby providing an effective tool for the efficient development and scientific verification of efficacy cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0078] The present application will be described in further detail below in conjunction with the accompanying drawings and preferred embodiments, but those skilled in the art will appreciate that the drawings are for illustrative purposes only and thus should not be taken as a limitation on the scope of the present disclosure. In addition, unless specifically stated otherwise, the drawings are not drawn to scale and are simply conceived to conceptually represent the composition or configuration of the described objects and can include exaggerated displays, and the drawings are not necessarily drawn to scale.

[0079] Figure 1 Flow chart of the method for evaluating the soothing and repairing effects of a cosmetic raw material on in vitro neurodermatitis.

[0080] Figure 2 Relative expression amount of neurogenic inflammatory response gene NK-1R and KRT14 in different groups of 3D skin models of neurodermatitis model constructed by capsaicin stimulation.

[0081] Figure 3 Relative expression amount of inflammatory genes IL-1β and TNF-α in different groups of 3D skin models of neurodermatitis model constructed by capsaicin stimulation.

[0082] Figure 4 Relative expression amount of skin barrier function genes FLG, LOR and CLDN1 in different groups of 3D skin models of neurodermatitis model constructed by capsaicin stimulation.

[0083] Figure 5 Relative secretion amount of neuropeptide factor substance P in different groups of 3D skin models of neurodermatitis model constructed by capsaicin stimulation.

[0084] Figure 6 Relative secretion amount of skin swelling and inflammatory factors IL-6 and CXCL8 in different groups of 3D skin models of neurodermatitis model constructed by capsaicin stimulation.

[0085] Figure 7 3D skin thickness in different groups of 3D skin models of neurodermatitis model constructed by capsaicin stimulation.

[0086] Figure 8 Transdermal water loss value of 3D skin in different groups of 3D skin models of neurodermatitis model constructed by capsaicin stimulation.

[0087] Figure 9 Relative fluorescence intensity of TRPV1 in different groups of 3D skin models of neurodermatitis model constructed by capsaicin stimulation.

[0088] Figure 10 Relative secretion amount of histamine in different groups of mast cells cultured by conditioned medium after capsaicin-induced neurodermatitis model.

[0089] Figure 11 Figure 1 is a diagram of the relative expression of NOS3 and ET-1 genes in different groups of blood vessel endothelial cells cultured in the conditioned medium after the construction of a model of neurodermatitis stimulated by capsaicin.

[0090] Figure 12 Figure 2 is a diagram of the relative secretion of VEGF in different groups of blood vessel endothelial cells cultured in the conditioned medium after the construction of a model of neurodermatitis stimulated by capsaicin. DETAILED DESCRIPTION

[0091] The following will be described in detail in combination with the accompanying drawings. Figures 1 to 12 The present application will be described in detail.

[0092] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described in detail in combination with examples. It should be understood that the specific examples described herein are only used to explain the present disclosure, and are not used to limit the present disclosure.

[0093] The experimental materials used in the embodiments of the present application are shown in Table 1.

[0094] Table 1 Experimental materials

[0095] Unless otherwise specified, the raw materials and materials used in the embodiments of the present application are purchased through general commercial channels.

[0096] The instruments used in the embodiments of the present application are shown in Table 2.

[0097] Table 2 Instruments

[0098] The sample of the raw material to be tested (test object) used in the embodiments of the present application is shown in Table 3.

[0099] Table 3 Sample of raw material to be tested

[0100] According to the literature

Zhou D X; Qian J; Li B Y; Wu J F; Deng P P. Comparison of soothing effects of dipotassium glycyrrhizinate [J]. Guangdong Chemical Industry, 2023, 50(17): 53-56.

Qiu H R. Synergistic anti-inflammatory study of bisabolol and gingerol [D]. Jiangnan University, 2008.

[0101] The present application is implemented as follows: Example 1 The present embodiment provides a method for constructing a neurodermatitis model stimulated by capsaicin, which is specifically as follows: 3D skin model pretreatment; EpiDerm TM The 3D skin model is placed in a 6-well plate for stable culture. 1ml of special culture medium is added to each well. The culture conditions are 37±5℃, (5±1)% CO2 and saturated humidity. The stable culture is carried out for 12h.

[0102] Mast cell inoculation and culture pretreatment; the mast cells are inoculated into a 6-well plate at a cell amount of 1*10^6-5*10^6 cells per well. The culture conditions are 37±5℃, (5±1)% CO2 and saturated humidity. The cells are cultured until they adhere and stably proliferate.

[0103] Vascular endothelial cell inoculation and culture pretreatment; the vascular endothelial cells are inoculated into a 6-well plate at a cell amount of 5*10^5-5*10^6 cells per well. The culture conditions are 37±5℃, (5±1)% CO2 and saturated humidity. The cells are cultured until they adhere and stably proliferate.

[0104] Take out the culture plate with the 3D skin model after cultivation and place it in a biological safety cabinet. Move the 3D skin model into a 6-well plate with fresh culture medium, set up three parallel groups, then add 1 mL of 100 μM concentration of capsaicin working solution to the 3D skin model. Incubate at 37 ± 5°C, (5 ± 1) % CO2 and saturated humidity for 12-48 h, and collect the incubated 3D skin model and the culture medium of the 3D skin model.

[0105] Mix the incubated 3D skin model culture medium with the mast cell culture medium at a ratio of 1:1 to prepare the mast cell conditioned medium. Discard the original culture medium in the hole plate of the pre-treated mast cells, and add 2 mL of the mast cell conditioned medium. Transfer the hole plate to the incubator and culture at 37 ± 5°C, (5 ± 1) % CO2 and saturated humidity for 24 hours. Collect the cultured mast cells and the culture medium of the mast cells.

[0106] Mix the incubated 3D skin model culture medium with the vascular endothelial cell culture medium at a ratio of 1:1 to prepare the vascular endothelial cell conditioned medium. Discard the original culture medium in the hole plate of the pre-treated vascular endothelial cells, and add 2 mL of the vascular endothelial cell conditioned medium. Transfer the hole plate to the incubator and culture at 37 ± 5°C, (5 ± 1) % CO2 and saturated humidity for 24 hours. Collect the cultured vascular endothelial cells and the culture medium of the vascular endothelial cells.

[0107] The neurodermatitis model stimulated by capsaicin was prepared by the above preparation method, and the model included the 3D skin model stimulated by capsaicin and incubated; the culture medium of the 3D skin model after being stimulated by capsaicin and incubated; The mast cells co-cultured with the culture medium of the 3D skin model after being stimulated by capsaicin and incubated and the culture medium of the mast cells after co-culture; The vascular endothelial cells co-cultured with the culture medium of the 3D skin model after being stimulated by capsaicin and incubated and the culture medium of the vascular endothelial cells after co-culture.

[0108] Example 2 The present embodiment provides a method for evaluating the in-vitro neurodermatitis soothing and repairing effect of a cosmetic raw material. The evaluation method of the present embodiment uses the model constructed in Example 1, as shown in Figure 1 Specifically as follows: S0: 3D skin model, mast cells, vascular endothelial cells, and pre-treatment of the sample to be tested; S0-1: Pre-treatment of the 3D skin model; place the EpiDerm TM Place the 3D skin model into a 6-well plate for stable cultivation. Add 1 ml of special culture medium to each hole. The cultivation conditions are 37 ± 5°C, (5 ± 1) % CO2 and saturated humidity, and the stable cultivation lasts for 12 h.

[0109] S0-2: Mast cell inoculation culture pretreatment; mast cells are inoculated into a 6-well plate at a cell amount of 1*10^6-5*10^6 cells per well, and the culture conditions are 37±5°C, (5±1)% CO2, and saturated humidity, and the cells are cultured until they adhere and stably proliferate.

[0110] S0-3: Vascular endothelial cell inoculation culture pretreatment; vascular endothelial cells are inoculated into a 6-well plate at a cell amount of 5*10^5-5*10^6 cells per well, and the culture conditions are 37±5°C, (5±1)% CO2, and saturated humidity, and the cells are cultured until they adhere and stably proliferate.

[0111] S0-4: Pretreatment of the sample to be tested; Pretreatment of the raw material sample A1 to be tested: Take an appropriate amount of the raw material sample A1 to be tested, use the culture medium as the solution, and prepare a working solution with a working concentration of 0.5%.

[0112] Pretreatment of the positive drug: Take an appropriate amount of the positive drug, dissolve it in dimethyl sulfoxide, vortex to fully dissolve it, filter it through a 0.22 μm filter membrane to remove bacteria, then divide and store it at -20°C in the dark to obtain a stock mother liquor. During the experiment, dilute the stock mother liquor with the culture medium to obtain a working solution with a working concentration of 100 μM. Throughout the process, ensure that the final concentration of dimethyl sulfoxide does not exceed 1% (v / v).

[0113] S1: Capsaicin stimulation of 3D skin model; take the 3D skin model pretreated before culture, and set up blank control group, negative control group, positive control group, and sample group; except for the blank control group, the negative control group, the positive control group, and the sample group each use 1 mL of 1 mM capsaicin working solution to stimulate and induce the 3D skin model pretreated before culture; place the 3D skin models in each group after stimulation and induction in the culture medium; set up three parallel groups for each group.

[0114] S2: 3D skin model drug treatment and culture incubation; the blank control group set in step S1' is not treated with drugs; fresh culture medium is added to the negative control group; 1 mL of 100 mM positive drug is added to each well of the positive control group; the sample group is added with the raw material sample A1 to be tested; then the 3D skin models in each group are cultured and incubated at a temperature of 37±5°C, (5±1)% CO2, and saturated humidity for 24 hours, and the 3D skin models and the culture medium of the 3D skin models after incubation in each group are collected.

[0115] S3: Detection of indicators in the 3D skin models and the culture medium of the 3D skin models after incubation and data processing; The expression amount of different genes in the 3D skin model cells after incubation, the secretion amount of different factors in the 3D skin model culture medium after incubation, the skin thickness of the 3D skin model after incubation, the transdermal water loss, and the protein expression amount are detected, and the relative expression amount of different genes in the 3D skin model cells in each group is calculated; the relative secretion amount of different factors in the 3D skin model culture medium in each group; the skin thickness, transdermal water loss value, and protein relative expression amount of the 3D skin model in each group; Specifically as follows: The indexes related to neurogenic inflammation, such as TRPV1, P substance, KRT14, and NK-1R, the indexes related to immune response and inflammatory reaction, such as IL-1β, IL-6, TNF-α, and CXCL8, the indexes related to skin barrier function, such as FLG, LOR, CLDN1, transdermal water loss value, and skin thickness, and the indexes related to vascular response, such as NOS3, ET-1, histamine, and VEGF, in each group of 3D skin models after incubation are detected. S3-1: Real-time fluorescent quantitative PCR is used to detect the gene expression amount of the 3D skin model cells after incubation. After incubation of the 3D skin model, the culture plate with the 3D skin model is taken out from the incubator, the 3D skin model is rinsed with DPBS buffer, the residual liquid on the surface of each 3D skin model is gently absorbed with a cotton swab, the 3D skin model is cut and transferred to a centrifuge tube, and the RNA in the skin tissue is extracted by using the Trizol method.

[0116] The extracted RNA is subjected to system preparation according to the kit instructions, and the reverse transcription operation is performed in a water bath at 37℃ for 15 min and at 85℃ for 5 s to obtain cDNA.

[0117] The PCR reaction solution is prepared according to a 20 μL system: 0.7 μL cDNA, 0.8 μL primer, 10 μL FastStart Essential DNA Green Master 2×conc, 8.5 μL H2O.

[0118] The reaction solution is added to a special 96-well plate and placed in a fluorescent quantitative PCR instrument for amplification reaction, and the instrument program is set as follows: Pre-incubation: 95℃ for 300 s, 1 cycle 3-Step Amplification: 95℃ for 10 s, 60℃ for 10 s, 72℃ for 10 s, 40 cycles Melting: 95℃ for 10 s; 65℃ for 60 s, 97℃ for 1 s, 1 cycle Cooling: 37℃ for 30 s, 1 cycle.

[0119] Obtain the Ct value of NK-1R, KRT14, IL-1β, TNF-α, FLG, LOR, CLDN1, NOS3, ET-1 corresponding to different samples.

[0120] S3-2: ELISA detection of factor secretion amount in 3D skin model culture solution after incubation; After incubation of the 3D skin model, the culture plate with the 3D skin model was taken out from the incubator, the culture solution in the well plate was blown evenly and then collected into a centrifuge tube, and the content of substance P, IL-6, CXCL8, VEGF, histamine was detected by ELISA kit. The operation steps are consistent with the kit instruction manual, and the optical density (Optical Density) value of substance P, IL-6, CXCL8, VEGF, histamine corresponding to different samples is obtained, which is abbreviated as OD value.

[0121] S3-3: H&E staining to detect the thickness of 3D skin model; The 3D skin was cut off with scissors and transferred to a centrifuge tube, 4% paraformaldehyde was added for fixation, and the 3D skin tissue was dehydrated with 50%-100% ethanol gradient, then liquid paraffin was added to the tube at 65℃ overnight, and then the skin tissue was embedded and solidified with paraffin. The embedded 3D skin tissue was sectioned and attached to a glass slide, and the glass slide was baked overnight using a slide baker. The glass slide was stained with hematoxylin and eosin, and then resin was used for sealing, and 7-10 images were taken under the microscope for each glass slide.

[0122] S3-4: Detection of trans-epidermal water loss of 3D skin model after incubation; The skin was taken out of the well plate, the surface liquid was wiped off with a cotton swab, and the skin was allowed to stand at room temperature for a certain period of time. After the skin was stable at room temperature, the trans-epidermal water loss detector was placed on top of the skin culture cup to form a sealed space, and the trans-epidermal water loss value (TEWL) was measured. Each skin model was measured 3-5 times.

[0123] S3-5: Immunofluorescence staining to detect protein expression in 3D skin model after incubation After the glass slide was baked overnight using a slide baker, the glass slide was subjected to antigen repair, blocking, first antibody (TRPV1) staining, second antibody staining, and nucleus staining in sequence. After staining, the glass slide was sealed with an anti-fluorescence quenching sealing agent, and 7-10 images were taken under different fields of view for each glass slide using a fluorescence microscope.

[0124] The relative expression of different genes in the cells of each group of 3D skin models after incubation was calculated respectively; the relative secretion of different factors in the culture medium of each group of 3D skin models after incubation was calculated; the skin thickness, trans-epidermal water loss value, and relative protein expression of each group of 3D skin models after incubation were calculated; The Ct data of different genes in the cells of each group of 3D skin models after incubation were collected, and 2^ The relative expression of the target gene was calculated by the method of ΔΔCt, and the calculation formula is as follows: ΔCt = Ct (target gene) - Ct (internal reference gene) ΔΔCt = ΔCt (test group) - ΔCt (negative control group) Relative expression ratio = 2^ ΔΔCt; The internal reference gene was GAPDH gene.

[0125] The relative secretion of different factors in the culture medium of each group of 3D skin models after incubation was calculated using the ELISA Calc regression / fitting calculation program to calculate the standard curve. The OD value of the test group was subtracted from the background hole OD value and then entered into the standard curve to calculate the content of the factor. The relative expression of inflammatory factors was calculated based on the negative control group as 100%, and the calculation formula is as follows: Relative expression = sample group content / negative control group content.

[0126] S4: Conditioned medium culture of mast cells; the culture medium of the 3D skin model after incubation was mixed with the mast cell culture medium at a ratio of 1:1 to prepare the mast cell conditioned medium. The culture medium of the 3D skin model after incubation was used to set up blank control group, negative control group, and positive control group. For the mast cells in the 6-well plate in S0-2, the original culture medium was discarded, and 2 mL of conditioned medium was added to each well according to different groups. The plate was transferred to the incubator and incubated at 37±5°C, 5%±1% CO2 and saturated humidity for 24-36 h. The cultured mast cells and culture medium of each group were collected.

[0127] S5: Detection of indicators in the culture medium of the cultured mast cells and data processing; The amount of histamine released in the culture medium of each group of cultured mast cells was detected using an ELISA kit, and the operation steps were strictly performed according to the kit instructions. The relative release of histamine in the culture medium of each group of cultured mast cells was calculated respectively; The OD values of the test groups were subtracted from the background hole OD values and then entered into the standard curve to calculate the content of the factor using the ELISA Calc regression / fitting calculation program. The relative expression amount of the inflammatory factor was calculated as 100% of the negative control group, and the calculation formula is as follows: Relative expression amount = sample group content / negative control group content.

[0128] S6: Conditioned medium culture of vascular endothelial cells; the incubated 3D skin model culture fluid was mixed with the vascular endothelial cell culture medium at a ratio of 1:1 to prepare the vascular endothelial cell conditioned medium. The incubated 3D skin model culture fluid was used to set up blank control group, negative control group and positive control group. For the vascular endothelial cells in S0-3 which had been stably cultured in 6-well plates, the original culture medium in the well plate was discarded, and 2 mL of conditioned medium was added to different groups, respectively. The well plate was transferred to the incubator and cultured at 37±5°C, (5±1)% CO2 and saturated humidity for 24-36h. The cultured vascular endothelial cells and the culture medium of the vascular endothelial cells were collected.

[0129] S7: Detection of indicators in cultured vascular endothelial cells and culture medium of vascular endothelial cells and data processing; The ELISA kit was used to detect the secretion amount of different factors in the culture medium of the cultured vascular endothelial cells, and the operation steps were strictly performed according to the kit instructions.

[0130] After the culture medium was collected, PBS was added to wash each well. 1 mL of Trizol was added to each well to blow the cells until the cells were completely detached from the well wall. The liquid was transferred to a centrifuge tube, and after RNA extraction, the expression of different genes in the cells was detected. The specific steps were consistent with S3-1 in S3, and the Ct values of different genes corresponding to each group were obtained.

[0131] Referring to step S3, the relative expression amount of different vascular reaction genes in the cultured vascular endothelial cells of each group was calculated; the relative secretion amount of different vascular reaction factors in the culture medium of the cultured vascular endothelial cells of each group was calculated; and the NOS3, ET-1 gene relative expression ratio = 2^ △△Ct; the relative expression amount of VEGF factor = sample group content / negative control group content.

[0132] S8: Result analysis and scoring; The relative expression amounts of NK-1R, KRT14, IL-1β, TNF-α, FLG, LOR, CLDN1, NOS3, ET-1 genes and the relative secretion amounts of substance P, IL-6, CXCL8, VEGF, histamine were plotted using GraphPad Prism, and the results were expressed as Mean ± SD. One-way ANOVA statistical analysis was used for comparison between groups. All statistical analyses were two-tailed. p<0.05 was considered to have a slight significant difference, represented by “*”; p<0.01 was considered to have a significant difference, represented by “**”; p<0.001 was considered to have a very significant difference, represented by “***”; p<0.0001 was considered to have an extremely significant difference, represented by “****”.

[0133] S9: comprehensive evaluation and efficacy evaluation; According to the weight distribution of the characteristic dimensions A to D, the scores of each index in the sample group were obtained by weighted comprehensive evaluation to obtain a comprehensive score, and the in-vitro neurodermatitis soothing and repairing efficacy of the cosmetic raw material was evaluated. The weight distribution of the characteristic dimensions A to D is as follows: Characteristic dimension A: the weight coefficient of the neurogenic inflammation characteristic dimension is 30%; Characteristic dimension B: the weight coefficient of the immune response and inflammation reaction characteristic dimension is 30%; Characteristic dimension C: the weight coefficient of the skin barrier function characteristic dimension is 25%; Characteristic dimension D: the weight coefficient of the vascular response characteristic dimension is 15%.

[0134] Examples 3-5 Examples 3, 4, and 5 differ from Example 2 in that the samples of the raw materials to be tested are different. The sample of the raw material to be tested in Example 3 is A2, the sample of the raw material to be tested in Example 4 is B1, and the sample of the raw material to be tested in Example 5 is B2.

[0135] The relative expression amounts of NK-1R, KRT14, IL-1β, TNF-α, FLG, LOR, CLDN1 genes in Examples 2-5 are shown in Figure 2 , Figure 3 , Figure 4 , wherein Figure 2 is the relative expression amount of NK-1R and KRT14 mRNA, Figure 3 is the relative expression amount of IL-1β and TNF-α mRNA, Figure 4 is the relative expression amount of FLG, LOR, and CLDN1 mRNA. Wherein *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 are the comparison results of the rest of the groups with the negative control group. From Figures 2-4It can be seen that the expression of NK-1R, KRT14, IL-1β, TNF-α genes in the negative control group is significantly higher than that in the blank control group, and the expression of FLG, LOR, CLDN1 genes is significantly down-regulated, indicating that the neurogenic inflammation and immune inflammation reaction is significantly enhanced after capsaicin induction treatment, and the skin barrier function is reduced; the expression of NK-1R, KRT14, IL-1β, TNF-α genes in the positive control group is significantly lower than that in the negative control group, and the expression of FLG, LOR, CLDN1 genes is significantly increased, indicating that the addition of positive drug capsaicinol significantly relieves the neurogenic inflammation and immune inflammation reaction, improves the skin barrier function, and the model is successfully constructed. After adding samples with soothing effect: sample A1, sample A2, sample B1, sample B2 can significantly reduce the expression of inflammation-related genes NK-1R, KRT14, IL-1β, TNF-α, and can improve the expression of barrier-related genes FLG, LOR, CLDN1 to different degrees, and the inhibition of inflammation and the soothing effect of barrier of sample A1 and A2, sample B1 and B2 are different.

[0136] The relative secretion amounts of P substance, IL-6 and CXCL8 are shown in Figure 5 and Figure 6 . Among them, Figure 5 is the relative secretion amount diagram of P substance, Figure 6 is the relative secretion amount diagram of IL-6 and CXCL8. Among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 is the comparison between sample group and negative control group. From Figure 5 and Figure 6 , it can be seen that the expression of P substance, IL-6 and CXCL8 in the negative control group is significantly higher than that in the blank control group, indicating that the neurogenic inflammation and immune inflammation reaction is significantly enhanced after capsaicin induction treatment; the expression of P substance, IL-6 and CXCL8 in the positive control group is significantly lower than that in the negative control group, indicating that the addition of positive drug capsaicinol significantly relieves the neurogenic inflammation and immune inflammation reaction, and the model is successfully constructed. After adding samples with soothing effect: sample A1, sample A2, sample B1, sample B2 can significantly reduce the expression of inflammation-related genes P substance, IL-6 and CXCL8, and the inhibition effect of sample A1 and A2, sample B1 and B2 on inflammation is different.

[0137] The skin thickness and trans-epidermal water loss value are shown in Figure 7 and Figure 8 . Among them, Figure 7 is the skin thickness statistical diagram, Figure 8 is the trans-epidermal water loss value diagram. Among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 is the comparison between sample group and negative control group. FromFigure 7 And Figure 8 It can be seen that the skin thickness of the negative control group is significantly lower than that of the blank control group, and the trans-epidermal water loss value is significantly increased, indicating that the skin barrier is damaged to a certain extent after capsaicin induction treatment; the skin thickness of the positive control group is significantly higher than that of the negative control group, and the trans-epidermal water loss value is significantly increased, indicating that the addition of the positive drug piperlongumine has a certain repair effect on the skin barrier, and the model is successfully constructed. After adding samples with soothing effect: sample A1, sample A2, sample B1, sample B2 can significantly increase the skin thickness and reduce the trans-epidermal water loss, and the repair efficacy of sample A1 and A2, sample B1 and B2 on the barrier is different.

[0138] The relative fluorescence intensity of TRPV1 is shown in Figure 9 . Among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 is the comparison between sample group and negative control group. It can be seen from Figure 9 that the relative fluorescence intensity of TRPV1 of the negative control group is higher than that of the blank control group, indicating that the neurogenic inflammation is up-regulated after capsaicin induction treatment; the addition of the positive drug reduces the relative fluorescence intensity of TRPV1, indicating that the positive drug can inhibit neurogenic inflammation to a certain extent, and the model is successfully constructed. After adding samples with soothing effect: sample A1, sample A2, sample B1, sample B2 can reduce the relative fluorescence intensity of TRPV1, and the soothing efficacy of sample A1 and A2, sample B1 and B2 on neurogenic inflammation is different.

[0139] The relative release amount of histamine in example 2-5 is shown in Figure 10 . Among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 is the comparison between sample group and negative control group. It can be seen from Figure 10 that the expression of histamine in the negative control group is significantly higher than that in the blank control group, indicating that the vascular response is significantly enhanced after capsaicin induction treatment; the expression of histamine in the positive control group is significantly lower than that in the negative control group, indicating that the addition of the positive drug piperlongumine significantly relieves the vascular response, and the model is successfully constructed. After adding samples with soothing effect: sample A1, sample A2, sample B1, sample B2 can significantly reduce the expression of histamine, and the inhibition efficacy of sample A1 and A2, sample B1 and B2 on vascular response is different.

[0140] The expression of vascular response related genes and factors in example 2-5 is shown in Figure 11 and Figure 12 . Among them Figure 11 is the NOS3 and ET-1 gene expression graph, Figure 12For the relative secretion amount of VEGF growth factor, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 is the comparison between sample group and negative control group. From Figure 11 and Figure 12 It can be seen that the expression of NOS3 in the negative control group is lower than that in the blank control group, and the expressions of ET-1 and VEGF are significantly increased, indicating that the vascular response is significantly enhanced after capsaicin induction treatment; the expression of NOS3 in the positive control group is higher than that in the negative control group, and the expressions of ET-1 and VEGF are significantly reduced, indicating that the addition of positive drug capsaicin significantly relieves the vascular response, and the model is successfully constructed. After adding samples with soothing effect: sample A1, sample A2 and sample B2 can significantly increase the expression of NOS3, sample A1, sample A2, sample B1 and sample B2 can significantly reduce the expressions of ET-1 and VEGF, and the inhibitory effect of sample A1 and A2 and sample B1 and B2 on vascular response is different.

[0141] Using the relative expression amount of NK-1R, KRT14, IL-1β, TNF-α, FLG, LOR, CLDN1, NOS3, ET-1 genes and the relative secretion amount of substance P, IL-6, CXCL8, VEGF, histamine, and using GraphPad Prism for plotting, the results are expressed as Mean±SD. One-way ANOVA statistical analysis was used for comparison between groups. All statistical analyses were two-tailed. p<0.05 is considered to have a slight significant difference, represented by “*”; p<0.01 is considered to have a significant difference, represented by “**”; p<0.001 is considered to have a very significant difference, represented by “***”; p<0.0001 is considered to have an extremely significant difference, represented by “****”.

[0142] According to the relative expression amount of NK-1R, KRT14, IL-1β, TNF-α, FLG, LOR, CLDN1 genes, the relative secretion amount of substance P, IL-6, CXCL8, the skin thickness, the trans-epidermal water loss value, the relative fluorescence intensity of TRPV1, the relative release amount of histamine, the relative expression amount of vascular response genes NOS3 and ET-1 genes, and the relative secretion amount of vascular response factor VEGF growth factor in each group of the above-mentioned embodiment 2, and the statistical analysis and scoring of each index, the expression of NK-1R, KRT14, IL-1β, TNF-α genes in the negative control group of embodiment 2 is obviously higher than that in the blank control group, the expression of FLG, LOR, CLDN1 genes is significantly down-regulated, the expression of substance P, IL-6, CXCL8 is obviously increased, and the relative fluorescence intensity of TRPV1 is increased. The skin thickness of the negative control group of embodiment 2 is obviously lower than that of the blank control group, and the trans-epidermal water loss value is significantly increased. The negative control group has skin barrier function damage in the characteristic dimension C. The expression of histamine in the negative control group of embodiment 2 is obviously higher than that in the blank control group, the expression of NOS3 is decreased, the expression of ET-1 and VEGF is obviously increased, and the negative control group has vascular response activation in the characteristic dimension D. At least one of the indexes in the negative control group of embodiment 2 has a significant difference compared with the blank control group, and the model of embodiment 2 is successfully constructed.

[0143] The expression of NK-1R, KRT14, IL-1β, TNF-α genes in the positive control group of embodiment 2 is obviously down-regulated compared with the negative control group, the expression of FLG, LOR, CLDN1 genes is obviously increased, the expression of substance P, IL-6, CXCL8 is obviously decreased, and the relative fluorescence intensity of TRPV1 is decreased. The positive control group of embodiment 2 has inhibited activation of neurogenic inflammation in the characteristic dimension A, and has inhibited activation of immune and inflammatory response in the characteristic dimension B. The skin thickness of the positive control group of embodiment 2 is obviously increased compared with the negative control group, and the trans-epidermal water loss value is obviously increased. The positive control group has repaired skin barrier function damage in the characteristic dimension C. The expression of histamine in the positive control group of embodiment 2 is obviously decreased compared with the negative control group, the expression of NOS3 is increased, the expression of ET-1 and VEGF is obviously decreased, and the positive control group has relieved vascular response activation in the characteristic dimension D. The model of embodiment 2 is effective.

[0144] The experimental significant difference results of the four samples in embodiment 2-5 in the 3D skin model are shown in table 4, and the evaluation and scoring are carried out according to table 5. The sample neurodermatitis soothing and repairing efficacy evaluation results are shown in table 6, and the final score of the sample comprehensive soothing and repairing efficacy evaluation is shown in table 7.

[0145] Table 4 Statistical difference results table of samples to be tested (test substances)

[0146] Table 5 Scoring rule table

[0147] Table 6 Test material score results

[0148] Table 7 Test material final score

[0149] According to the above results, the soothing repair redness efficacy of the four samples is ranked as follows: Sample A2 > Sample A1, Sample B2 > Sample B1.

[0150] Therefore, the final results of the construction and scoring system of the model are highly consistent with the human patch test results reported in the above literature, which is consistent with the human patch test results. It is shown that the use of the model for evaluating the soothing effect of cosmetic products and raw materials can save time and cost of human experiments, and can more efficiently and accurately obtain experimental results consistent with human experiments. The construction method of the capsaicin stimulation to construct the neurodermatitis model in the present application at least covers a multi-dimensional joint evaluation system of sensitive skin core phenotypes such as nerves, inflammation, barrier, and vascular response. The multi-index comprehensive evaluation based on the weight coefficient can effectively solve the bottleneck of efficacy evaluation in the development of soothing repair products or raw materials, and provide a multi-dimensional system evaluation standard for the soothing repair efficacy verification and evaluation of cosmetic products, which can be effectively used for the soothing repair efficacy strength and concentration screening of cosmetic raw materials, and provide effective reference for product development and efficacy verification.

[0151] Comparative Examples 1-4 The difference between Comparative Example 1 and Example 2, Comparative Example 2 and Example 3, Comparative Example 3 and Example 4, and Comparative Example 4 and Example 5 lies in the different weight distribution coefficients of the characteristic dimensions, and the weight coefficients of the four characteristic dimensions of Comparative Examples 1-4 are the same: Characteristic dimension A: the weight coefficient of the neurogenic inflammation characteristic dimension is 25%; Characteristic dimension B: the weight coefficient of the immune response and inflammation reaction characteristic dimension is 25%; Characteristic dimension C: the weight coefficient of the skin barrier function characteristic dimension is 25%; Characteristic dimension D: the weight coefficient of the vascular response characteristic dimension is 25%.

[0152] According to the average distribution of the multi-dimensional weight, the final scores of the test raw material samples A1, A2, B1, and B2 are recalculated as shown in Table 8, and the soothing repair efficacy of the test raw material samples is evaluated.

[0153] Table 8 Final score of test raw material sample after average distribution of weight

[0154] According to the above results, the soothing repair redness efficacy of the two groups of samples is ranked as follows: Sample A1 > Sample A2, Sample B2 > Sample B1.

[0155] Therefore, the final results of the model evaluation of Comparative Examples 1-4 are not completely consistent with the human patch results, and do not completely conform to the human patch experiment results, indicating that the average allocation of weights is not applicable to the evaluation method of the in vitro neurodermatitis soothing repair efficacy of cosmetic raw materials.

[0156] Examples 6-12 On the basis of Example 2, the present application carried out implementation research on different 3D skin models, different capsaicin working solution concentrations and amounts, different capsaicin working solution concentrations and amounts, and different ratios and amounts of conditioned medium of co-cultured mast cells and vascular endothelial cells. The specific parameters are shown in Table 9. According to Table 9, each of the sample groups of Examples 6-12 is provided with 4 sample groups.

[0157] Table 9 Research parameters of Examples 6-12

[0158] The present application detected, analyzed and scored each index of Examples 6-12. The negative control group of Examples 4-10 showed characteristic dimension A neurogenic inflammation activation, characteristic dimension B immune response and inflammation reaction activation, characteristic dimension C skin barrier function impairment, and characteristic dimension D vascular response activation. The index scores of the negative control group had a significant difference from the index scores of the blank control group. In each of the examples, the positive control group showed a change trend opposite to that when the model was successfully constructed, and the models of Examples 6-12 were all successfully constructed.

[0159] On the basis of Example 2, the present application evaluated the in vitro neurodermatitis soothing repair efficacy of the test raw material samples A1, A2, B1 and B2 using the models of Examples 6-12. The ranking of the samples of Examples 6-12 was consistent with the evaluation results of Examples 2-5, i.e., Sample A2 > Sample A1, and Sample B2 > Sample B1.

[0160] Therefore, EpiSkin TM , SkinEthic TM RHE, T-Skin TM 3D skin model, EpiDerm TM 3D skin model can be applicable to the evaluation of the in vitro neurodermatitis soothing repair efficacy of cosmetic raw materials.

[0161] Based on the embodiment 2, the application provides a screening method of a cosmetic raw material with neurodermatitis soothing and repairing effect. The neurodermatitis soothing and repairing effect of a to-be-tested raw material sample is evaluated by using the evaluation method of the cosmetic raw material in the embodiment 2, and a raw material showing the neurodermatitis soothing and repairing effect is selected.

[0162] The application is described in detail above, and the principles and implementation modes of the application are described by using specific examples. The above description of the examples is only used to help understand the disclosure and the core idea. It should be pointed out that, for ordinary skilled persons in the technical field, some improvements and modifications can be made to the application without departing from the principles of the application, and these improvements and modifications also fall within the protection scope of the claims of the application.

Claims

1. A neurodermatitis model constructed with stimulation of capsaicin, characterized by, The model comprises: a 3D skin model stimulated by capsaicin and incubated; a culture solution of the 3D skin model after being stimulated by capsaicin and incubated; a mast cell co-cultured with the culture solution of the 3D skin model after being stimulated by capsaicin and incubated and a culture solution of the mast cell after co-culturing; a vascular endothelial cell co-cultured with the culture solution of the 3D skin model after being stimulated by capsaicin and incubated and a culture solution of the vascular endothelial cell after co-culturing.

2. The neurodermatitis model using capsaicin stimulation according to claim 1, wherein The model is used to evaluate neurodermatitis; the neurodermatitis has one or more detectable characteristic dimensions in characteristic dimensions A-D: Characteristic dimension A: neurogenic inflammatory response characteristic dimension; Characteristic dimension B: immune response and inflammatory response characteristic dimension; Characteristic dimension C: skin barrier function characteristic dimension; Characteristic dimension D: vascular response characteristic dimension; The 3D skin model stimulated by capsaicin and incubated is used to reflect one or more of characteristic dimension A, characteristic dimension B, characteristic dimension C and characteristic dimension D of the neurodermatitis; The culture solution of the 3D skin model after being stimulated by capsaicin and incubated is used to reflect one or more of characteristic dimension A, characteristic dimension B and characteristic dimension D of the neurodermatitis; The culture solution of the mast cell after being co-cultured with the culture solution of the 3D skin model after being stimulated by capsaicin and incubated is used to reflect characteristic dimension D of the neurodermatitis; The culture solution of the vascular endothelial cell after being co-cultured with the culture solution of the 3D skin model after being stimulated by capsaicin and incubated is used to reflect characteristic dimension D of the neurodermatitis.

3. The neurodermatitis model using capsaicin stimulation according to claim 2, wherein Characteristic dimension A is embodied by one or more of the following indexes: up-regulation of expression amount of nerve sensitivity and perception related genes; the nerve sensitivity and perception related genes include one or more of TRPV1, TRPV2, TRPA1, ASIC3 and SCN9A; up-regulation of expression amount of neuropeptide and neuropeptide related receptor genes; the neuropeptide includes one or more of substance P, CGRP, VIP and NPY; the neuropeptide related receptor genes include one or more of substance P receptor NK-1R and CGRPR; up-regulation of secretion amount of neuropeptide related factors; the neuropeptide related factors include one or more of substance P, CGRP, VIP and NPY factors; up-regulation of expression amount of keratinization related genes; the keratinization related genes include one or more of KRT1, KRT10, KRT14, KRT16, KRT17 and KLK5; up-regulation of expression amount of TRP receptor family proteins; the TRP receptor family proteins include one or more of TRPV1 and TRPA1; up-regulation of expression amount of neuropeptide related receptor proteins; the neuropeptide related receptor proteins include one or more of NK-1R and CGRPR proteins; Characteristic dimension B is embodied by one or more of the following indexes: up-regulation of expression amount of inflammatory response related genes; the inflammatory response related genes include one or more of IL-1α, IL-1β, IL-6, IL-10, IL-12β, IL-12, IL-17, IL-22, IL-33, TNF-α and CXCL8; up-regulation of expression amount of inflammatory response related genes; the inflammatory response related genes include one or more of IL-1α, IL-1β, IL-6, IL-10, IL-12β, IL-12, IL-17, IL-22, IL-33, TNF-α and CXCL8; The expression amount of the immune regulation related genes is up-regulated, and the immune regulation related genes include one or more of TLR2, TLR4, TLR9, NFKB1 and NFKB2; The secretion amount of the skin swelling and inflammation related factors is up-regulated, and the skin swelling and inflammation related factors include one or more of IL-1α, IL-1β, IL-6, CXCL8, IL-12, IL-17, IL-22, IL-33 and TNF-α factors; The characteristic dimension C is embodied by one or more of the following indexes: The expression amount of the skin barrier structure protein genes is down-regulated, and the skin barrier structure protein genes include one or more of FLG, LOR, IVL, TGM1, CLDN1, OCLN, DSP, DSG, ZO-1 and SPINK5; The expression amount of the dry desquamation related protease and inhibitor genes is up-regulated, and the dry desquamation related protease and inhibitor genes include one or more of KLK7, MMP9 and MMP1; The skin physiological function index; the skin physiological function index includes one or more of the following: the trans-epidermal water loss value is significantly increased, and the epidermal thickness is significantly reduced; The expression amount of the skin barrier structure protein is down-regulated, and the skin barrier structure protein includes one or more of FLG, LOR, IVL and DSG protein; The expression amount of the tight junction protein is down-regulated, and the tight junction protein includes one or more of CLDN1 and ZO-1 protein; The characteristic dimension D is embodied by one or more of the following indexes: The amount of histamine released in the mast cell culture solution after co-culture is up-regulated; The expression amount of the vascular response related genes is down-regulated, and the vascular response related genes include one or more of ETBR, VEGFR2, TIE2, PECAM-1, ICAM-1, VCAM-1, NOS3 and ET-1 genes; The secretion amount of the vascular response related factors is up-regulated, and the vascular response related factors include one or more of NO, ET-1, ICAM-1 and VCAM-1 factors; The secretion amount of the vascular response related growth factors is up-regulated, and the vascular response related growth factors include one or more of VEGF, EGF, FGF and PDGF factors.

4. The method for constructing a neurodermatitis model using capsaicin stimulation according to claim 1, wherein The method comprises the following steps: The 3D skin model is stimulated by using a capsaicin working solution, and the stimulated 3D skin model is incubated in a culture solution, and the incubated 3D skin model and the culture solution of the 3D skin model are collected; The culture solution of the incubated 3D skin model is prepared into a mast cell conditioned medium by using a mast cell culture medium, the mast cell is cultured by using the mast cell conditioned medium, and the cultured mast cell and the mast cell culture solution are collected; The culture solution of the incubated 3D skin model is prepared into a vascular endothelial cell conditioned medium by using a vascular endothelial cell culture medium, the vascular endothelial cell is cultured by using the vascular endothelial cell conditioned medium, and the cultured vascular endothelial cell and the vascular endothelial cell culture solution are collected.

5. The method for constructing a neurodermatitis model using capsaicin stimulation according to claim 4, wherein the capsaicin is applied to the skin of the subject in an amount of 0.01 to 0.1 mg / cm2. said 3D skin model is selected from the group consisting of a human recombinant 3D epidermis model or a human recombinant 3D full skin model; including EpiSkin TM , SkinEthic TM RHE, T-Skin TM , EpiDerm TM 3D skin model; The working concentration of capsaicin is 0.1-3mM; wherein, EpiSkin TM The working concentration of capsaicin in 3D skin model is 2-3mM; SkinEthic TM The working concentration of capsaicin in RHE 3D skin model is 0.1-0.3mM; T-Skin TM The working concentration of capsaicin in 3D skin model is 3-5mM; EpiDerm TM The working concentration of capsaicin in 3D skin model is 1-2mM; The incubation conditions of the stimulated 3D skin model, the culture conditions of the mast cell and the vascular endothelial cell are all 37±5℃, (5±1)% CO2 and saturated humidity. ​ The incubation time of the 3D skin model after stimulation is 12-48 hours; the culture time of mast cells and vascular endothelial cells is 24-36 hours; The volume ratio of the culture solution of the incubated 3D skin model to the mast cell culture medium or the vascular endothelial cell culture medium is 1: (1-3).

6. A method for evaluating the soothing and repairing efficacy of a cosmetic raw material on neurodermatitis in vitro, using the neurodermatitis model constructed using capsaicin stimulation according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: S1: Take the 3D skin model, and set up a blank control group, a negative control group, a positive control group, and a sample group; except the blank control group, the negative control group, the positive control group, and the sample group are stimulated and induced by using a capsaicin working solution on the 3D skin model; and then the 3D skin model of each group after the stimulation and induction is placed in a culture solution; S2: The blank control group set in step S1 is not subjected to drug addition; fresh culture medium is added to the negative control group; positive drugs are added to the positive control group; and the sample group is added with a sample to be tested; and then the 3D skin model of each group is cultured and incubated; and the 3D skin model of each group after the incubation and the culture solution of the 3D skin model of each group are collected; S3: The expression amount of different genes in the 3D skin model cells after the incubation, the secretion amount of different factors in the culture solution of the 3D skin model after the incubation, the skin thickness, the transdermal water loss, and the protein expression amount of the 3D skin model of each group after the incubation are detected; the relative expression amount of different genes in the 3D skin model cells of each group is calculated; the relative secretion amount of different factors in the culture solution of the 3D skin model of each group is calculated; the skin thickness, the transdermal water loss, and the relative protein expression amount of the 3D skin model of each group are calculated; S4: Conditioned medium is used to culture mast cells; The culture solution of the 3D skin model of each group after the incubation collected in step S2 is prepared into a mast cell conditioned medium by being mixed with a mast cell culture medium; the mast cell conditioned medium is used to culture mast cells; and the mast cells of each group after the culture and the culture solution of the mast cells are collected; S5: The histamine release amount in the culture solution of the mast cells of each group after the culture in step S4 is detected; and the relative release amount of histamine in the culture solution of the mast cells of each group after the culture is calculated; S6: Conditioned medium is used to culture vascular endothelial cells; The culture solution of the 3D skin model of each group after the incubation collected in step S2 is prepared into a vascular endothelial cell conditioned medium by being mixed with a vascular endothelial cell culture medium; the vascular endothelial cell conditioned medium is used to culture vascular endothelial cells; and the vascular endothelial cells of each group after the culture and the culture solution of the vascular endothelial cells are collected; S7: The expression amount of different genes in the vascular endothelial cells of each group after the culture in step S6 is detected; the secretion amount of different factors in the culture solution of the vascular endothelial cells of each group after the culture is detected; the relative expression amount of different genes in the vascular endothelial cells of each group after the culture is calculated; and the relative secretion amount of different factors in the culture solution of the vascular endothelial cells of each group after the culture is calculated; S8: The relative expression amount of different genes in the 3D skin model cells after the incubation, the relative secretion amount of different factors in the culture solution of the 3D skin model after the incubation, the relative protein expression amount, the skin thickness, and the transdermal water loss of each group in step S3 are subjected to statistical analysis. Statistical analysis was performed on the relative release amount of histamine in the culture solution of each group of mast cells after culture in step S5. Statistical analysis was performed on the relative expression amount of different genes and the relative secretion amount of different factors in the blood vessel reaction of each group after culture in step S7. Each detection index of the sample group was scored according to the difference between the sample group and the negative control group in the corresponding detection index. S9: Comprehensive evaluation of the scores of each detection index in the sample group was performed to evaluate the in-vitro neurodermatitis soothing and repairing efficacy of the cosmetic raw material.

7. The method for evaluating soothing and repairing efficacy on neurodermatitis of the cosmetic raw material in vitro according to claim 6, wherein the cosmetic raw material is applied to the skin of the subject. According to the difference between the sample group and the negative control group in the corresponding detection index, each detection index of the sample group was scored. The scoring criteria were as follows: p<0.05, slight significant difference, 0.3 points; p<0.01, significant difference, 0.5 points; p<0.001, very significant difference, 0.7 points; P<0.0001, extremely significant difference, 1.0 points; Comprehensive evaluation of the scores of each detection index in the sample group was performed to evaluate the in-vitro neurodermatitis soothing and repairing efficacy of the cosmetic raw material. The comprehensive score was obtained by weighted comprehensive evaluation of the scores of each index in the sample group according to the weight distribution of characteristic dimensions A to D, and the in-vitro neurodermatitis soothing and repairing efficacy of the cosmetic raw material was evaluated. The weight distribution of characteristic dimensions A to D was as follows: Characteristic dimension A: the weight coefficient of the neurogenic inflammation characteristic dimension was 30%; Characteristic dimension B: the weight coefficient of the immune response and inflammation reaction characteristic dimension was 30%; Characteristic dimension C: the weight coefficient of the skin barrier function characteristic dimension was 25%; Characteristic dimension D: the weight coefficient of the blood vessel reaction characteristic dimension was 15%.

8. The method for evaluating soothing and repairing efficacy on neurodermatitis of the cosmetic raw material in vitro according to claim 6, wherein, The positive drug is capsaicin; the working solution concentration of the positive drug is 40-1000 μM; wherein, EpiSkin TM 3D skin model The working solution concentration of the positive drug in the model is 400-600 μM; SkinEthic TM 3D skin model The working solution concentration of the positive drug in the model is 400-600 μM; SkinEthic TM 3D skin model The working solution concentration of the positive drug in the model is 400-600 μM; SkinEthic TM 3D skin model The working solution concentration of the positive drug in the model is 400-600 μM; SkinEthic 9. A screening method for cosmetic raw materials having soothing and repairing efficacy for neurodermatitis, characterized by, The screening method of the cosmetic raw material used the evaluation method of the in-vitro neurodermatitis soothing and repairing efficacy of the cosmetic raw material according to any one of claims 6-8 to evaluate the neurodermatitis soothing and repairing efficacy of the raw material sample to be tested, and selected the raw material showing the neurodermatitis soothing and repairing efficacy.

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