A daily-use preservative skin comprehensive toxicity prediction and verification method based on cuticle-fibroblast co-culture multi-pathway response

Abstract

This invention discloses a method for predicting and validating the comprehensive skin toxicity of daily-use preservatives based on multi-pathway responses in keratinocyte-fibroblast co-culture, belonging to the technical field of daily chemical safety evaluation. This invention uses human keratinocytes (HaCaT) and human dermal fibroblasts (HSF) as dual targets, and assesses the mechanistic skin toxicity of preservatives through multiple endpoints including inflammation, barrier damage, cell senescence, and organelle damage, classifying them and guiding their formulation application. The method of this invention boasts high prediction accuracy, high sensitivity, high efficiency, and compliance with regulations. It overcomes the limitations of traditional methods that only focus on "cell viability," providing a panoramic analysis of the toxic action pathways of preservatives from three dimensions: gene expression (early signal), organelle damage (intermediate process), and population apoptosis (final outcome), avoiding false negatives caused by single endpoints and possessing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of safety evaluation technology for daily chemical products. Specifically, it relates to a method for evaluating the mechanistic skin toxicity of preservatives and classifying them into grades, and guiding their formulation application, by using human keratinocytes (HaCaT) and human dermal fibroblasts (HSF) as dual targets and through multiple endpoints such as inflammation, barrier damage, cell senescence, and organelle damage. Background Technology

[0002] Preservatives are an indispensable ingredient in daily cosmetics, but their potential skin irritation and toxicity have always been a focus of industry attention. Traditional preservative skin toxicity testing mainly relies on animal Draize assays or single in vitro cell viability tests (such as the MTT assay). However, animal testing faces ethical controversies, long cycles, and significant species variability, and is restricted by regulations in many countries. Existing in vitro alternatives, such as reconstructed epidermal models (OECD TG439, ​​TG492), while reducing animal use, typically use tissue viability (mitochondrial dehydrogenase activity) as the sole endpoint.

[0003] This binary (dead / alive) evaluation model has significant flaws: First, it cannot distinguish the specific forms of cell death (such as apoptosis, necroptosis, autophagy, pyroptosis, etc.), and because the skin immune responses induced by different death modes are quite different, mechanistic information is missing; second, the sensitivity of a single endpoint is insufficient, and it cannot capture the sublethal damage (such as barrier protein degradation, inflammatory factor release, organelle stress, etc.) caused by long-term exposure to preservatives at low doses, resulting in a serious underestimation of the cumulative risk of "long-term exposure to low doses".

[0004] Therefore, developing an evaluation method that can mimic the skin's keratinocyte-dermis interaction and analyze toxicity mechanisms from multiple dimensions, including gene expression, cell population phenotype, and subcellular organelle microstructure, is of great significance for guiding the development of mild preservative formulations that achieve "reduced toxicity without reduced efficacy". Summary of the Invention

[0005] To address the issue that existing skin toxicity methods only provide binary information on "tissue or cell death-life," and cannot analyze which cell death / inflammatory pathways preservatives trigger adverse skin reactions at low exposure doses (≤IC20).

[0006] The primary objective of this invention is to provide a method for predicting and validating the comprehensive skin toxicity of daily preservatives based on multi-pathway responses of keratinocyte-fibroblast co-culture. The specific method includes:

[0007] (1) Cell model construction and exposure: Human keratinocytes and human dermal fibroblasts were mixed and cultured. After the culture was completed, the preservative solution to be tested was added and exposed to culture to obtain exposed cells;

[0008] (2) Gene transcription level detection (Gene-Score): Total RNA was extracted from the exposed cells, and qPCR was used to detect the gene expression levels covering apoptosis, necrosis, autophagy, pyroptosis, inflammation, and barrier-related pathways, and the gene score (Gene-Score) was calculated.

[0009] (3) Cell population phenotypic detection (Apop-Score): The exposed cells were subjected to double staining, and the total apoptosis rate was detected by flow cytometry to obtain the apoptosis score (Apop-Score).

[0010] (4) Subcellular organelle structure and function detection (Org-Score): The exposed cells were subjected to fluorescence treatment, and the mitochondrial membrane potential, superoxide level and lysosomal state were quantified by high-content imaging to obtain the organelle score (Org-Score).

[0011] (5) Scoring and evaluation: The gene score, apoptosis score and organelle score are integrated to obtain the comprehensive toxicity score (Tox-Score), and the toxicity level is classified according to the comprehensive toxicity score.

[0012] Optionally, in some embodiments, the ratio of the number of human keratinocytes to the number of human dermal fibroblasts in step (1) is (1.5-3):1.

[0013] Preferably, the ratio of the number of human keratinocytes to the number of human dermal fibroblasts is 2:1.

[0014] Optionally, in some embodiments, the human keratinocytes and human dermal fibroblasts in step (1) are both in the logarithmic growth phase, and the total seeding amount of the human keratinocytes and human dermal fibroblasts is (0.8-2) × 10⁻¹⁰. 5 cells / pores.

[0015] Human keratinocytes and human dermal cells were chosen as cell models in this invention because the skin is composed of the epidermis (mainly keratinocytes) and the dermis (mainly fibroblasts), and there is complex paracrine signaling communication between the two. If the proportion of keratinocytes is too high, the supporting role of the dermis in the epidermal barrier and the feedback effect of amplified inflammation cannot be reflected; if the proportion of fibroblasts is too high, it will mask the actual exposure scenario where the preservative first comes into contact with the epidermis. This invention preferably uses a 2:1 co-culture ratio, which can better simulate the physiological structural characteristics of the human skin's "epidermis-dermis" and ensure the authenticity of the toxic response.

[0016] Optionally, in some embodiments, the conditions for the mixed culture in step (1) are: temperature 36-38℃; CO2 concentration 3-7%; time 20-28h.

[0017] Optionally, in some embodiments, the solvent of the preservative solution to be tested includes 0.2% BSA and 1% bispecific antibody in DMEM high sugar, the inoculum amount of the preservative solution to be tested is 10^5 / well, and the concentration of the preservative solution to be tested is set as needed.

[0018] Optionally, in some embodiments, the exposure culture conditions in step (1) are: temperature 36-38℃; CO2 concentration 3-7%; time 20-28h.

[0019] In the method for predicting and verifying the comprehensive skin toxicity of preservatives in this invention, if the exposure time is less than 18 hours, the changes in the transcriptional level of some genes have not yet been converted into changes in the protein level or organelle morphology, which may easily lead to false negatives; if the exposure time exceeds 30 hours, cells may experience secondary necrosis or non-specific nutrient deficiency, which may interfere with the toxicity assessment.

[0020] Optionally, in some embodiments, the total RNA extraction method in step (2) includes the Trizol method.

[0021] Optionally, in some embodiments, the apoptotic gene in step (2) includes at least one of Bax, Bcl-2, Caspase-3, and Caspase-9.

[0022] Optionally, in some embodiments, the necrotic gene in step (2) includes at least one of RIPK1, RIPK3, and MLKL.

[0023] Optionally, in some embodiments, the genes involved in the autophagy-related pathway in step (2) include MTOR and / or BECN1.

[0024] Optionally, in some embodiments, the genes involved in the pyroptosis-related pathway in step (2) include at least one of GSDMD, IL-18, and IL-1β.

[0025] Optionally, in some embodiments, the genes of the inflammation-related pathways described in step (2) include NLRP3 and / or IL-6.

[0026] Optionally, in some embodiments, the genes of the barrier-related pathways described in step (2) include FLG and / or LOR.

[0027] Optionally, in some embodiments, the gene score (Gene-Score) calculation method in step (2) includes: using β-actin as an internal reference, calculating the relative expression level through 2^-ΔΔCt to obtain the gene score Gene-Score (log2FC average).

[0028] Existing cell proliferation and detection technologies (MTT) only provide information on cell viability. This invention, by screening key genes in the aforementioned apoptosis, necrosis, autophagy, pyroptosis, inflammation, and barrier-related pathways, can distinguish whether a preservative induces "quiet" apoptosis (Caspase lineage), or triggers strong inflammatory responses such as necrosis (RIPK lineage) or pyroptosis (GSDMD lineage), or disrupts the physical barrier (FLG / LOR), thereby achieving precise tracing of the toxic mechanism.

[0029] Optionally, in some embodiments, step (3) includes the double staining process of: digesting the exposed cells with trypsin, washing them with PBS solution, and then performing double staining with Annexin V-FITC / PI.

[0030] Optionally, in some embodiments, in step (3), the total apoptosis rate is the apoptosis score (Apop-Score).

[0031] Optionally, in some embodiments, in step (4), the mitochondrial membrane potential state includes the mitochondrial membrane potential loss rate (%ΔΨm loss).

[0032] Optionally, in some embodiments, in step (4), the superoxide level status includes the superoxide positivity rate (%MitoSOX+).

[0033] Optionally, in some embodiments, in step (4), the lysosomal status includes the lysosomal deformity rate (%Lysotracker+).

[0034] Optionally, in some embodiments, in step (4), the organelle score (Org-Score) is the arithmetic mean of the mitochondrial membrane potential loss rate, the superoxide positivity rate, and the lysosomal abnormality rate.

[0035] Optionally, in some embodiments, in step (5), the comprehensive toxicity score is the arithmetic mean of three scores: gene transcription level detection (Gene-Score), cell population phenotype detection (Apop-Score), and subcellular organelle structure and function detection (Org-Score). That is, Tox-Score = (Gene-Score + Apop-Score + Org-Score) / 3.

[0036] Optionally, in some embodiments, in step (5), the correspondence between the comprehensive toxicity score and the toxicity level is as follows: Level I (≤0.20) is safe and non-toxic; Level II (0.20–0.40) is mildly toxic; Level III (0.40–0.60) is moderately toxic; Level IV (0.60–0.80) is highly toxic; and Level V (>0.80) is severely toxic.

[0037] Another objective of this invention is to provide an application of a method for predicting and validating the comprehensive skin toxicity of daily preservatives based on multiple pathway responses of keratin-fibroblast co-culture in the in vitro toxicity evaluation of daily chemical preservatives.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] (1) Comprehensive mechanism analysis: This invention breaks through the limitations of traditional methods that only focus on "cell life and death". It provides a panoramic analysis of the toxic action pathway of preservatives from three dimensions: gene expression (early signal), organelle damage (intermediate process) to population apoptosis (final outcome), avoiding false negatives caused by a single endpoint.

[0040] (2) High prediction accuracy: The keratin-fibroblast co-culture model is adopted, which preserves the communication network between skin cells. The experimental results show that the Tox-Score of this invention has a correlation of up to 0.85 with historical Draize animal test data, which is far superior to single cell models or single detection methods.

[0041] (3) High sensitivity: It can identify sublethal damage at low concentrations (≤IC20), especially the early risks caused by mitochondrial ROS accumulation or inflammatory gene upregulation, providing a screening basis for "mild preservative" formulations.

[0042] (4) High efficiency and compliance: The testing cycle only takes 5 days, and all testing indicators have commercially available standardized reagent kits. The data output conforms to the 3R principle (reduce, replace, and optimize animal experiments), which is conducive to standardization and promotion. Attached Figure Description

[0043] Figure 1 The experimental flowchart of the multidimensional evaluation method provided in the embodiments of the present invention is shown.

[0044] Figure 2 This is a bar chart showing gene expression after phenoxyethanol treatment in an embodiment of the present invention.

[0045] Figure 3 The images show flow cytometry apoptosis detection and statistical graphs of phenoxyethanol-treated cells in this embodiment of the invention. Detailed Implementation

[0046] The present invention will be further described below with reference to embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments can be obtained commercially.

[0047] Unless otherwise specified, all percentages mentioned in this invention are percentages by mass.

[0048] The statistical analysis of the method for predicting and verifying the comprehensive skin toxicity of daily preservatives of the present invention was performed using IBM SPSS Statistical 19. *P<0.05 was significant, and **P<0.01 was extremely significant.

[0049] The primer sequences for gene transcription involved in this invention are shown in Table 1.

[0050] Table 1 Primer sequence listing

[0051]

[0052] The human keratinocytes and human dermal fibroblasts of this invention are derived from Suzhou Haixing Biotechnology Co., Ltd.

[0053] The culture medium formula for the mixed culture of human keratinocytes and human dermal fibroblasts in step (1) of this invention is as follows: by volume percentage, each cell culture medium contains 10% fetal bovine serum (FBS), 1% streptomycin-penicillin and 89% DMEM high glucose medium.

[0054] The formulation of the culture medium for exposure culture in step (1) of this invention is as follows: by volume percentage, each part of the exposure culture medium contains 0.2% bovine serum albumin (BSA), 1% streptomycin, 1% penicillin, and 97.8% DMEM high-glucose medium. Different preservative concentrations are prepared by first preparing a 0.5% stock solution using the exposure culture medium as a solvent and then diluting it stepwise.

[0055] The instruments involved in this invention include: QuantStudio 6 real-time fluorescence PCR instrument (Thermofisher, USA); 5825 low-temperature high-speed centrifuge (Eppendorf, Germany); Cytation 3 multi-functional microplate reader (BioTek, USA); MX204 / A electronic analytical balance (Mettler Toledo, Germany); and LSM500 laser confocal microscope (ZEISS, Germany).

[0056] Example 1: Comprehensive toxicity assessment of phenoxyethanol.

[0057] according to Figure 1The evaluation process shown is used for multidimensional biological evaluation.

[0058] (1) Cell model construction and exposure: HaCaT (human keratinocytes) in logarithmic growth phase were mixed with HSF (human dermal fibroblasts, also known as HDF) at a ratio of 2:1. DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin was used. Cells were seeded in 96-well plates (1×10^4 cells / well), 24-well plates (5×10^4 cells / well), and 6-well plates (1×10^5 cells / well) and cultured at 37℃ and 5% CO2 for 24 h until adherence. The old medium was removed, and fresh exposure solution containing different concentrations of phenoxyethanol was added (solvent was DMEM high-glucose medium containing 0.2% BSA and 1% penicillin). The phenoxyethanol concentration gradient was set at 0% (blank control), 0.05%, 0.1%, and 0.2% (w / v). The cells were exposed for another 24 hours to obtain the exposed cells.

[0059] (2) Gene transcription level detection (obtaining Gene-Score): Cells were collected from 6-well plates, and total RNA was extracted using the Trizol method, followed by reverse transcription to obtain cDNA. To balance mechanism tracing and the 3R parsimony principle, six core genes validated by the chemical training set were selected for qPCR detection: apoptosis (Caspase-3), necrosis (RIPK3), autophagy (mTOR), pyroptosis (GSDMD), inflammation (NLRP3), and barrier (FLG). β-actin was used as an internal reference to calculate Log2FC. The calculation formula was: Gene-Score = mean(log2FC_g) for g ∈ {Caspase-3, RIPK3, BECN1, GSDMD, NLRP3, FLG}, with the result rounded to two decimal places.

[0060] (3) Cell population phenotypic detection (obtaining the Apop-Score): Cells were collected from 6-well plates, digested with trypsin, washed with PBS, and then subjected to Annexin V-FITC / PI double staining. Flow cytometry was used to collect 10,000 events, and the percentage of cells in the Q2 (late apoptosis) + Q3 (early apoptosis) regions was calculated to obtain the apoptosis score (Apop-Score). The calculation formula was: Apop-Score = (Events_Q2 + Events_Q3) / 10,000 × 100%, with the mean of 3 replicates taken and rounded to one decimal place.

[0061] (4) Subcellular organelle structure and function detection (Obtaining Org-Score): Cells in 24-well plates were fixed with 4% paraformaldehyde and incubated with Mito-Tracker 488 (labeled mitochondria), MitoSOX Red (labeled mitochondrial superoxide), and Lysotracker Deep Red (labeled lysosomes) working solutions for 30 minutes in the dark. High-content imaging systems were used to photograph cells under a 20× objective lens, and the fluorescence intensity of single cells was automatically analyzed to calculate the Org-Score. Org-Score = (MFI_ %ΔΨmloss + MFI_%MitoSOX+ + MFI_ %Lysotracker Deep Red ) / 3, retaining one decimal place; negative values ​​were truncated to 0%.

[0062] (5) Scoring and evaluation: The arithmetic mean of the gene transcription level detection (Gene-Score) in step (2), the cell population phenotype detection (Apop-Score) in step (3), and the subcellular organelle structure and function detection (Org-Score) was calculated to obtain the comprehensive toxicity score (Tox-Score). The calculation formula is Tox-Score = (Gene-Score + Apop-Score + Org-Score) / 3. Based on the correspondence between the comprehensive toxicity score and the toxicity level in Table 2, the toxicity relationship of phenoxyethanol at different concentrations was obtained. The relevant results are shown in Table 3.

[0063] Table 2. Correspondence between comprehensive toxicity score and toxicity level

[0064]

[0065] Table 3 Calculation results of phenoxyethanol concentrations in Example 1

[0066]

[0067] Conclusion: In this embodiment, the linear correlation coefficient r between the full gradient (0–0.2%) three-segment Tox-Score and historical Draize skin irritation scores was 0.85 (P<0.01), meeting the OECD TG 492 requirement for "high in vivo correlation" for in vitro alternative methods. Figure 2 In this study, gene expression of BCL-2, Caspase-3, RIPK3, MTOR, GSDMD, NLRP3, and LOR was analyzed. Phenoxyethanol significantly upregulated core genes of the death pathway at a 0.1% level (Gene-Score 0.68). Figure 3In this study, cell mortality was detected by flow cytometry. At the population level, approximately 40% of cells had entered late / early apoptosis (Apop-Score 38.3%). At the subcellular organelle level, decreased mitochondrial membrane potential, superoxide accumulation, and increased lysosomal acidity were observed (Org-Score 21.4%), indicating significant perturbation of the gene-death-organelle cascade. It is recommended that the formulation use a concentration <0.05%. Under this concentration, long-term use of phenoxyethanol does not pose a cumulative risk (such as barrier protein degradation, inflammatory factor release, organelle stress, etc.). The safety threshold or effective concentration of phenoxyethanol reported in existing studies (such as Macova et al., 2012 toxicological evaluation of aquatic organisms and Corsini et al., 2016 assessment of skin sensitization risk) is around 0.05%, which is highly consistent with the detection results in Example 1 of this study. However, the aforementioned studies primarily rely on live animal models such as the zebrafish embryo acute toxicity test (OECD 236) or large-scale data simulations, which not only face increasingly serious ethical controversies but also have limitations such as long evaluation cycles (usually 7-14 days) and low human relevance due to species differences. In contrast, the method developed in this study significantly shortens the evaluation cycle to 5 days, is simple to operate, and is compatible with commercially available standardized reagent kits, exhibiting excellent universality. Furthermore, the detection indicators of this evaluation system are highly aligned with current technical guidelines for in vitro alternative methods, and its data can directly support the registration of alternative methods for cosmetic raw materials.

[0068] A comparative test was conducted using phenoxyethanol at a concentration of 0.2%.

[0069] Comparative Example 1: Single-cell model (HaCaT only)

[0070] The only difference from Example 1 is that HSF cells are not added in step (1), and only HaCaT cells are seeded. The remaining detection steps are the same.

[0071] Comparative Example 2: Single-cell model (HSF only)

[0072] The only difference from Example 1 is that HaCaT cells are not added in step (1), only HSF cells are seeded. The remaining detection steps are the same.

[0073] Comparative Example 3: Single Detection Dimension (Gene Testing Only)

[0074] The only difference from Example 1 is that only step (2) gene transcription level detection (obtaining Gene-Score) is performed, and Gene-Score is used as the comprehensive toxicity score.

[0075] Comparative Example 4: Single Detection Dimension (Cell Population Phenotypic Detection Only)

[0076] The only difference from Example 1 is that only step (3) cell population phenotyping (obtaining Apop-Score) is performed, and Apop-Score is used as the comprehensive toxicity score.

[0077] Comparative Example 5: Single Detection Dimension (Subcellular Organelle Structure and Function Detection Only)

[0078] The only difference from Example 1 is that only step (4) subcellular organelle structure and function detection (obtaining Org-Score) is performed, and Org-Score is used as the comprehensive toxicity score.

[0079] Comparative Example 6: Two detection dimensions (gene detection and cell population phenotypic detection)

[0080] The only difference from Example 1 is that step (2) gene transcription level detection (obtaining Gene-Score) and step (3) cell population phenotype detection (obtaining Apop-Score) are performed, and the arithmetic mean of Gene-Score and Apop-Score is used as the comprehensive toxicity score.

[0081] Comparative Example 7: Two detection dimensions (gene detection and subcellular organelle structure and function detection)

[0082] The only difference from Example 1 is that step (2) gene transcription level detection (obtaining Gene-Score) and step (4) subcellular organelle structure and function detection (obtaining Org-Score) are performed, and the arithmetic mean of Gene-Score and Org-Score is used as the comprehensive toxicity score.

[0083] Comparative Example 8: Two detection dimensions (cell population phenotype detection and subcellular organelle structure and function detection)

[0084] The only difference from Example 1 is that step (3) cell population phenotype detection (obtaining Apop-Score) and step (4) subcellular organelle structure and function detection (obtaining Org-Score) are performed, and the arithmetic mean of Apop-Score and Org-Score is used as the comprehensive toxicity score.

[0085] The test results for Comparative Examples 1-8 are shown in Table 4.

[0086]

[0087] Comparing Example 1 with Comparative Examples 1 and 2, the Tox-Score of the HaCaT / HSF co-culture model was significantly higher than that of the single-cell model (0.53±0.32, 0.58±0.12). This indicates that a single cell type cannot fully simulate the complex response of skin tissue to preservatives. The co-culture model, through the interaction between keratinocytes and fibroblasts, more realistically reflects the toxic effects at the tissue level.

[0088] Comparative Example 5 (subcellular organelle structure and function detection only) is limited by the observation of a single physical phenotype, resulting in a high threshold for recognizing minute damage and a rating of Grade II, which easily leads to the omission of early biochemical damage. In contrast, Example 1 combines early warning data at the gene transcription level, sensitively capturing compensatory changes in organelles and accurately classifying them as Grade IV, effectively avoiding missed detections.

[0089] Comparative Example 3 (gene detection only) showed a Gene-Score as high as 1.24, but due to the excessive sensitivity of this indicator, the Tox-Score was artificially inflated, resulting in a Grade V rating. Fluctuations in gene expression often precede substantial damage, and relying solely on gene upregulation may lead to misjudgments of toxicity at low concentrations of substances. Example 1 introduced cell population apoptosis (57.5%) and organelle indicators for weighted evaluation, using the "late outcome" of the physical phenotype to logically calibrate the "early warning" of transcriptional levels, thus bringing the evaluation results back to a Grade IV that better reflects biological reality.

[0090] As the number of detection dimensions increased (e.g., compared to Comparative Examples 6-8 versus Comparative Examples 3-5), the correlation coefficient between the evaluation results and actual toxicity gradually improved, but none reached the level of full-dimensional detection in Example 1. This demonstrates that the three-stage evaluation system of "gene (early warning) - organelle (mid-term damage) - population apoptosis (late outcome)" has a significant synergistic effect. This full-chain evidence matrix from molecular to phenotype can effectively capture toxicity characteristics missed by single methods through the complementarity and mutual verification of multiple indicators, thereby completing a high-precision and high-reliability safety evaluation within 5 days.

Claims

1. A method for predicting and validating the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture, characterized in that, Includes the following steps: (1) Cell model construction and exposure: Human keratinocytes and human dermal fibroblasts were mixed and cultured. After the culture was completed, the preservative solution to be tested was added and exposed to culture to obtain exposed cells; (2) Gene transcription level detection: Total RNA was extracted from the exposed cells, and qPCR was used to detect the gene expression levels covering apoptosis, necrosis, autophagy, pyroptosis, inflammation, and barrier-related pathways, and gene scores were calculated; (3) Cell population phenotype detection: The exposed cells were subjected to double staining, and the total apoptosis rate was detected by flow cytometry to obtain an apoptosis score; (4) Subcellular organelle structure and function detection: The exposed cells were subjected to fluorescence treatment, and the mitochondrial membrane potential, superoxide level and lysosomal state were quantified by high-content imaging to obtain organelle scores; (5) Scoring and evaluation: The gene score, the apoptosis score and the organelle score are integrated to obtain a comprehensive toxicity score, and the toxicity level is classified according to the comprehensive toxicity score.

2. The method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture according to claim 1, characterized in that, The ratio of human keratinocytes to human dermal fibroblasts in step (1) is (1.5-3):

1.

3. The method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture according to claim 1, characterized in that, In step (1), both the human keratinocytes and the human dermal fibroblasts are in the logarithmic growth phase, and the total seeding amount of the human keratinocytes and the human dermal fibroblasts is (0.8-2) × 10⁻¹⁰. 5 cells / pores; The conditions for mixed culture described in step (1) are: temperature 36-38℃; CO2 concentration 3-7%; time 20-28h; The solvent of the preservative solution to be tested in step (1) includes 0.2% BSA and 1% double antibiotic DMEM high sugar, the inoculum amount of the preservative solution to be tested is 10^5 / well, and the concentration of the preservative solution to be tested is set according to the requirements; The conditions for exposure culture described in step (1) are: temperature 36-38℃; CO2 concentration 3-7%; time 20-28h.

4. The method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture according to claim 1, characterized in that, The apoptosis genes mentioned in step (2) include at least one of Bax, Bcl-2, Caspase-3, and Caspase-9; The necrotic genes mentioned in step (2) include at least one of RIPK1, RIPK3, and MLKL; The genes involved in the autophagy-related pathways described in step (2) include MTOR and / or BECN1; The genes involved in the pyroptosis-related pathway in step (2) include at least one of GSDMD, IL-18, and IL-1β; The genes involved in the inflammation-related pathways described in step (2) include NLRP3 and / or IL-6; The genes involved in the barrier-related pathways described in step (2) include FLG and / or LOR.

5. The method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture according to claim 1, characterized in that, The total RNA extraction method described in step (2) is the Trizol method; The gene score calculation method in step (2) includes: using β-actin as an internal reference, calculating the relative expression level through 2^-ΔΔCt to obtain the gene score.

6. The method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture according to claim 1, characterized in that, In step (3), the double staining process includes: digesting the exposed cells with trypsin, washing them with PBS solution, and then performing double staining with Annexin V-FITC / PI. In step (3), the total apoptosis rate is the apoptosis score.

7. The method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture according to claim 1, characterized in that, In step (4), The mitochondrial membrane potential state includes the mitochondrial membrane potential loss rate; The superoxide level status includes the superoxide positivity rate; The lysosomal status includes the lysosomal deformity rate; The organelle score is the arithmetic mean of the mitochondrial membrane potential loss rate, the superoxide positivity rate, and the lysosomal abnormality rate.

8. The method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture according to claim 1, characterized in that, In step (5), the comprehensive toxicity score is the arithmetic mean of the gene transcription level detection, cell population phenotype detection, and subcellular organelle structure and function detection.

9. The method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on multi-pathway response of keratin-fibroblast co-culture according to claim 1, characterized in that, In step (5), the correspondence between the comprehensive toxicity score and the toxicity level is as follows: Level I, ≤0.20, is safe and non-toxic; Level II, 0.20–0.40, is mildly toxic; Level III, 0.40–0.60, is moderately toxic; Level IV, 0.60–0.80, is highly toxic; Level V, >0.80, is severely toxic.

10. The application of the method for predicting and verifying the comprehensive skin toxicity of daily preservatives based on the multi-pathway response of keratin-fibroblast co-culture as described in claim 1 in the in vitro toxicity evaluation of daily chemical preservatives.

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