Establishment method and application of PC12 cell model for evaluating soothing efficacy of cosmetics

Abstract

The invention relates to a method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics. The method comprises the following steps: S1, determining model stimulation conditions; S11, detecting cell viability of a control group and cell viability of capsaicin groups with different concentrations by using a CCK-8 method; s12, observing the activation condition of the capsaicin on the TRPV1 channel of the PC12 cell by using a fluorescence microscope; s13, detecting the expression condition of the capsaicin to the TRPV1 of the PC12 cell by using an ELISA method; s14, detecting the expression condition of the capsaicin on the PC12 cell neuropeptide P substance by using an ELISA (enzyme-linked immuno sorbent assay) method; and S15, detecting the expression condition of capsaicin on IL-33 related to PC12 cell neuroinflammation by using an ELISA method. The invention also relates to evaluating the soothing efficacy of the cosmetic raw material. According to the method, the nerve-like cells are adopted to establish the cell model, the process that human skin is subjected to external stimulation and nerve reaction is excessive is effectively simulated, and the established in-vitro cell model can be used for evaluating the effectiveness of cosmetic raw materials / finished products in protecting or repairing skin nerve disorder and evaluating whether the cosmetic raw materials / finished products have soothing efficacy or not.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular to a method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics and an application thereof. Background Art

[0002] According to the "Chinese Expert Consensus on Sensitive Skin Diagnosis and Treatment", 40%-56% of women in Asia have sensitive skin, of which about 36.1% of women in my country have sensitive skin. China has become a country with a large population of sensitive skin and the number of sensitive people is increasing, which has driven the continuous expansion of the overall sensitive skin market. Many factors can induce or aggravate sensitive skin. Physical factors such as temperature and humidity changes, sunlight exposure, etc. will affect the symptoms of sensitive skin. Increased temperature and humidity and sun exposure will make the skin more sensitive. In addition, some chemical stimuli such as cosmetics, cleaning products, air pollutants, etc. will also aggravate sensitivity. In addition to various external factors, there are also many internal factors, such as genetics, gender, hormone levels, emotional mental state, age, etc.

[0003] The research on soothing ingredients can start from the mechanism of sensitive skin. Under the interaction of internal and external factors, the skin barrier function of sensitive skin is damaged, which causes an increase in sensory nerve afferent signals, thereby increasing the skin's reactivity to external stimuli and causing inflammatory reactions in the skin. The above causes often promote each other and accumulate, eventually leading to a vicious cycle. Since sensitive skin can cause various sensory symptoms, such as burning, stinging, itching, etc., it indicates that abnormal nerve sensory function in the skin may be one of the pathological mechanisms of sensitive skin. The occurrence of sensitive skin is related to the sensory nerve fibers in the epidermis. After the integrity of the skin barrier is damaged, the skin nerve endings are not adequately protected, exposing the skin nerve fiber endings to a stimulating environment, thereby causing an increase in the skin's sensory response. Sensitive skin patients have neuropathic pain and changes in epidermal nerve endings, which indicate that sensitive skin has a neurogenic sensitivity mechanism.

[0004] In studies investigating sensitive skin, most experiments have only studied the expression and downstream pathways of TRPV1 in keratinocytes. Zhou et al. (Citrus fruit extract regulates TRPV1 function to treat sensitive skin, Cosmet Journal of Dermatology, 2023.22(4): pp. 1369-1376) constructed a recombinant hTRPV1 high-expression cell line (HaCaT-TRPV1-OE cells) and screened substances and extracts from various plants. Li et al. (Inhibition of TRPV1 can prevent skin irritation caused by phenoxyethanol, International Journal of Cosmetic Science, 2017.39(1): pp. 11-16) used HaCaT cells cultured in vitro to explore whether TRPV1 is involved in the itching and burning sensation caused by phenoxyethanol. However, Pecze et al. compared the amount of mRNA in cultured keratinocytes, human HaCaT cells, and human trigeminal ganglion samples. The results showed that the level of TRPV1 transcripts in keratinocytes was several orders of magnitude lower than that in trigeminal ganglia. It is uncertain whether the cascade reaction of TRPV1 in keratinocytes is similar to that of nerves. After the skin receives various stimuli, it can produce many neuropeptides. They are mainly produced by neurons containing sensory nerve C fibers, bind to corresponding receptors, produce a series of biological reactions, and are transmitted to the central nervous system through nerves to form various sensations.

[0005] Nowadays, the research on skin barrier and inflammation is relatively mature, while the research on neural response is relatively rare. At this stage, most of the research is still focused on studying the abnormalities of the skin's neural sensory function using keratinocytes, but skin cells cannot simulate the response of nerves to stimulation. Summary of the invention

[0006] The present invention provides a method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics and its application. The constructed in vitro cell model can be used to evaluate the effectiveness of cosmetic raw materials / finished products in protecting or repairing skin nerve disorders. Nerve cells can better simulate the process of excessive nerve reaction and evaluate whether the cosmetic raw materials / finished products have soothing efficacy.

[0007] The technical solution of the present invention:

[0008] A method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics comprises the following steps: S1, determining the model stimulation conditions:

[0009] S11. Determine the effect of capsaicin treatment on the activity of PC12 highly differentiated cells: Use CCK-8 method to detect the cell viability of the control group and different concentrations of capsaicin groups;

[0010] S12. Determine the effect of capsaicin treatment on the activation of TRPV1 channels in PC12 cells: Use a fluorescence microscope to observe the activation of TRPV1 channels in PC12 cells by capsaicin;

[0011] S13. Determine the effect of capsaicin treatment on the expression of TRPV1 in PC12 cells: ELISA was used to detect the effect of capsaicin on the expression of TRPV1 in PC12 cells;

[0012] S14. Determine the effect of capsaicin treatment on the expression of substance P in PC12 cells: ELISA was used to detect the effect of capsaicin on the expression of neuropeptide substance P in PC12 cells;

[0013] S15. Determine the effect of capsaicin treatment on the expression of IL-33 in PC12 cells: Use ELISA to detect the effect of capsaicin on the expression of neuroinflammation-related IL-33 in PC12 cells.

[0014] Preferably, the method further comprises S2, determining a positive control:

[0015] S21. Determine the effect of positive substance treatment on the activity of PC12 highly differentiated cells: Use CCK-8 method to detect the cell viability of the control group and the positive substance groups with different concentrations;

[0016] S22. Determine the effect of positive substance treatment on the activation of TRPV1 channels in PC12 cells: Use a fluorescence microscope to observe the activation of TRPV1 channels in PC12 cells by positive substances;

[0017] S23. Determine the effect of positive substance treatment on the expression of TRPV1 in PC12 cells: ELISA method was used to detect the effect of positive substance treatment on the expression of TRPV1 in PC12 cells;

[0018] S24. Determine the effect of positive substance treatment on the expression of neuropeptide substance P in PC12 cells: ELISA method is used to detect the effect of positive substance treatment on the expression of neuropeptide substance P in PC12 cells;

[0019] S25. Determine the effect of positive substance treatment on the expression of IL-33 in PC12 cells: Use ELISA to detect the effect of positive substance treatment on the expression of neuroinflammation-related IL-33 in PC12 cells.

[0020] Preferably, the specific step of S11 is: taking PC12 cells in good growth state, and 1×10 5 cells / mL were inoculated in a 96-well plate; the groups were set as BC blank group, NC control group, and CA capsaicin group. After culturing for 24 hours, 100 μL of capsaicin solution was added to the BC and NC groups, and different concentrations of capsaicin solution, including 20-160 μmol / L, were added to the CA group; after continuing to culture for 24 hours, the CCK-8 reagent was operated according to the instructions.

[0021] Preferably, the cell viability (%) of the capsaicin group: [OD 450 (CA)-OD450 (BC) / OD 450 (NC)-OD 450 (BC)]×100%

[0022] OD 450 (CA) is the absorbance of the capsaicin-treated cell group at 450 nm, OD 450 (BC) is the absorbance of the complete culture medium group at 450 nm, OD 450 (NC) is the absorbance of the cell group treated with complete medium at 450 nm.

[0023] Preferably, the specific step of S12 is: taking PC12 cells in good growth state, at a rate of 1.5×10 5 The cells were inoculated in a 6-well plate at a density of 1.5 cells / mL. The groups were set as NC control group and CA capsaicin group. After culturing for 24 hours, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations, including 20-100 μmol / L, was added to the CA group. After culturing for another 24 hours, the cells were washed twice with HBSS solution, and Fluo-4, AM working solution was added to the cells. The cells were cultured at 37°C for 30 minutes, washed twice with HBSS, and cultured at 37°C for 10 minutes. The cells were immediately photographed under a fluorescence microscope.

[0024] Preferably, Fluo-4, AM working solution: Fluo-4, AM is dissolved in anhydrous DMSO to prepare a 2 mmol / L storage solution, 50 μg Fluo-4, AM is dissolved in 22.791 μL anhydrous DMSO; the Fluo-4, AM solution is diluted with basal culture medium to prepare a 4 μmol / L Fluo-4, AM working solution.

[0025] Preferably, the specific step of S13 is: taking PC12 cells in good growth state, at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC control group and CA capsaicin group. After culturing for 24 hours, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations, including 20-100 μmol / L, were added to the CA group. After culturing for another 24 hours, the cells were washed once with PBS, 200 μL of cell lysis buffer was added, and the cells were lysed on ice for 30 min-1 h. The expression of TRPV1 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0026] Preferably, the specific step of S14 is: taking PC12 cells in good growth state, at 1.5×10 5The cells were inoculated in a 6-well plate at a density of cells / mL, and the groups were set as NC control group and CA capsaicin group; after culturing for 24 hours, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations, including 20-100 μmol / L, and the cells were cultured for another 24 hours, then washed once with PBS, 200 μL of PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments for two times to destroy the cell membrane, the cell lysate was centrifuged at 2-8°C and 5000×g for 5 minutes to obtain the supernatant, which was used for subsequent ELISA and BCA experiments. The expression of substance P was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0027] Preferably, the specific step of S15 is: taking PC12 cells in good growth state, at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC control group and CA capsaicin group. After culturing for 24 hours, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations, including 20-100 μmol / L, were added to the CA group. After culturing for another 24 hours, the cells were washed once with PBS, 200 μL of PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments for two times to destroy the cell membrane, the cell lysate was centrifuged at 2-8°C and 5000×g for 5 minutes to obtain the supernatant, which was used for subsequent ELISA and BCA experiments. The ELISA kit was used to detect the expression of IL-33, and the BCA protein quantification kit was used for protein quantification.

[0028] The invention discloses an application of a method for establishing a PC12 cell model for evaluating the soothing efficacy of cosmetics, and assessing the soothing efficacy of cosmetic raw materials.

[0029] Beneficial effects of the present invention:

[0030] PC12 cells are a cell line differentiated from rat adrenal medullary pheochromocytoma. PC12 cell line is the preferred model for neurobiological research because it has neurogenic characteristics (after differentiation), such as neurotransmitter secretion (dopamine, norepinephrine and other catecholamines), neuronal morphology, and ion and neurotransmitter receptors. Capsaicin is a TRPV1 agonist that has the effect of regulating TRPV1 sensitivity. It induces a large amount of Ca by lowering the response threshold of TRPV1. 2+ Influx of capsaicin causes hyperalgesia, which eventually leads to increased nerve reactivity. Based on PC12 highly differentiated cells, a cell injury model of capsaicin damage was established to simulate the process of excessive skin nerve reaction and evaluate whether the cosmetic raw materials / finished products have soothing efficacy.

[0031] The present invention uses neuron-like cells to establish a cell model, which effectively simulates the process of excessive nerve response when human skin is stimulated by external stimuli. The constructed in vitro cell model can be used to evaluate the effectiveness of cosmetic raw materials / finished products in protecting or repairing skin nerve disorders, and to evaluate whether the cosmetic raw materials / finished products have soothing efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a graph showing the effect of different concentrations of capsaicin on the activity of PC12 cells after 24 hours of treatment.

[0033] Figure 2 This graph shows the effect of different concentrations of capsaicin on the activation of TRPV1 channels in PC12 cells after 24 hours of treatment.

[0034] Figure 3 This is a graph showing the effect of different concentrations of capsaicin on the expression of TRPV1 in PC12 cells after 24 hours of treatment.

[0035] Figure 4 This is a graph showing the effect of different concentrations of capsaicin on the expression of substance P in PC12 cells after 24 hours of treatment.

[0036] Figure 5 This is a graph showing the effect of different concentrations of capsaicin on the expression of IL-33 in PC12 cells after 24 hours of treatment.

[0037] Figure 6 This is a graph showing the effect of different concentrations of positive substances on the viability of PC12 cells after 24 hours of treatment.

[0038] Figure 7 This is a graph showing the effect of positive substance treatment on the activation of TRPV1 channels in PC12 cells for 24 hours.

[0039] Figure 8 This is a graph showing the effect of positive substance treatment on the expression of TRPV1 in PC12 cells for 24 hours.

[0040] Fig. 9 This is a graph showing the effect of positive substance treatment on the expression of substance P in PC12 cells for 24 hours.

[0041] Fig.10 This is a graph showing the effect of positive substance treatment on the expression of IL-33 in PC12 cells for 24 hours.

[0042] Fig.11 This is a graph showing the effect of different concentrations of sample 1 on the viability of PC12 cells after 24 hours of treatment.

[0043] Fig.12 This is a graph showing the effect of sample 1 on the expression of TRPV1 in PC12 cells after 24 hours of treatment.

[0044] Fig.13This is a graph showing the effect of different concentrations of sample 2 on the viability of PC12 cells after treatment for 24 hours.

[0045] Fig.14 This is a graph showing the effect of sample 2 on the expression of TRPV1 in PC12 cells after 24 hours of treatment.

[0046] Fig.15 This is a graph showing the effect of different concentrations of sample 3 on the viability of PC12 cells after treatment for 24 hours.

[0047] Fig.16 This is a graph showing the effect of sample 3 on the expression of TRPV1 in PC12 cells after 24 hours of treatment.

[0048] Fig.17 This is a graph showing the effect of different concentrations of sample 4 on the viability of PC12 cells after treatment for 24 hours.

[0049] Fig.18 This graph shows the effect of sample 4 on the expression of TRPV1 in PC12 cells after 24 hours of treatment. DETAILED DESCRIPTION

[0050] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation method, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0051] Example 1

[0052] 1. Determination of Model Stimulation Conditions

[0053] 1.1 Effect of capsaicin treatment on the activity of highly differentiated PC12 cells: The CCK-8 method was used to detect the cell viability of the control group and groups with different concentrations of capsaicin.

[0054] Specific operation: Take PC12 cells with good growth status and add 1×10 5 The cells were inoculated in a 96-well plate at a density of 10 cells / mL. The groups were set as BC (blank group), NC (control group), and CA (capsaicin group), with 6 parallels in each group. After 24 hours of culture, 100 μL of capsaicin solution was added to the BC and NC groups, and different concentrations of capsaicin solution (20, 40, 60, 80, 100, 120, 140, 160 μmol / L) were added to the CA group. After another 24 hours of culture, the CCK-8 reagent was operated according to the instructions.

[0055] The complete culture medium is: 89% by mass of RPMI1640 cell culture medium + 10% by mass of fetal bovine serum + 1% by mass of penicillin-streptomycin, the same below.

[0056] Cell viability in capsaicin group (%): [OD 450 (CA)-OD450 (BC) / OD 450 (NC)-OD 450 (BC)]×100%

[0057] OD 450 (CA) is the absorbance of the capsaicin-treated cell group at 450 nm, OD 450 (BC) is the absorbance of the complete culture medium group at 450 nm, OD 450 (NC) is the absorbance of the cell group treated with complete medium at 450 nm.

[0058] 1.2 Effect of capsaicin treatment on the activation of TRPV1 channels in PC12 cells: Fluorescence microscopy was used to observe the effects of capsaicin on the activation of TRPV1 channels in PC12 cells.

[0059] Solution preparation:

[0060] Fluo-4, AM working solution: Dissolve Fluo-4, AM in anhydrous DMSO to prepare a 2 mmol / L stock solution (dissolve 50 μg Fluo-4, AM in 22.791 μL anhydrous DMSO), dilute the Fluo-4, AM solution with basal culture medium to prepare a 4 μmol / L Fluo-4, AM working solution.

[0061] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC (control group) and CA (capsaicin group). After 24 hours of culture, 2 mL of capsaicin solution (20, 40, 60, 80, 100 μmol / L) was added to the NC group and 2 mL of capsaicin solution (20, 40, 60, 80, 100 μmol / L) was added to the CA group. After 24 hours of culture, the cells were washed twice with HBSS solution, and Fluo-4, AM working solution was added to the cells, cultured at 37°C for 30 minutes, washed twice with HBSS, and cultured at 37°C for 10 minutes to ensure the complete deesterification of AM bodies in the cells, and photographed immediately under a fluorescence microscope.

[0062] 1.3 Effect of capsaicin treatment on TRPV1 expression in PC12 cells: ELISA was used to detect the effect of capsaicin on TRPV1 expression in PC12 cells.

[0063] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC (control group) and CA (capsaicin group). After 24 hours of culture, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations (20, 40, 60, 80, 100 μmol / L) was added to the CA group. After 24 hours of culture, PBS was added to wash once, 200 μL of cell lysis solution was added, and lysis was carried out on ice for 30 min-1 h. The expression of TRPV1 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0064] 1.4 Effect of capsaicin treatment on the expression of substance P in PC12 cells: ELISA was used to detect the effect of capsaicin on the expression of neuropeptide substance P in PC12 cells.

[0065] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC (control group) and CA (capsaicin group). After 24 hours of culture, 2 mL of capsaicin solution of different concentrations (20, 40, 60, 80, 100 μmol / L) was added to the NC group and 2 mL of capsaicin solution of different concentrations (20, 40, 60, 80, 100 μmol / L) was added to the CA group. After 24 hours of continuous culture, the cells were washed once with PBS, 200 μL of PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments to destroy the cell membrane twice, the cell lysate was centrifuged at 5000 × g for 5 minutes at 2-8°C to obtain the supernatant. The supernatant was used for subsequent ELISA and BCA experiments. The expression of substance P was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0066] 1.5 Effect of capsaicin treatment on IL-33 expression in PC12 cells: ELISA was used to detect the effect of capsaicin on the expression of neuroinflammation-related IL-33 in PC12 cells.

[0067] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC (control group) and CA (capsaicin group). After 24 hours of culture, 2 mL of capsaicin solution of different concentrations (20, 40, 60, 80, 100 μmol / L) was added to the NC group and 2 mL of capsaicin solution of different concentrations (20, 40, 60, 80, 100 μmol / L) was added to the CA group. After 24 hours of continued culture, PBS was added to wash once, 200 μL PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments to destroy the cell membrane, the cell lysate was centrifuged at 2-8°C and 5000 × g for 5 minutes to obtain the supernatant. The supernatant was used for subsequent ELISA and BCA experiments. The expression of IL-33 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0068] 2. Determination of positive control

[0069] 2.1 Effect of positive substance treatment on the activity of PC12 highly differentiated cells: The CCK-8 method was used to detect the cell viability of the control group and the positive substance groups with different concentrations.

[0070] Specific operation: Take PC12 cells with good growth status and add 1×10 5 The cells were inoculated in a 96-well plate at a density of 1.5 cells / mL. The groups were set as BC (blank group), NC (control group), and PC (positive group). The BC group was added with 100 μL of complete culture, and the NC and PC groups were added with 100 μL of cell suspension, with 6 parallels in each group. After 24 hours of culture, 100 μL of complete culture was added to the BC and NC groups, and different concentrations of positive solution (0.5, 1, 2, 3, 4, 5, 6, 7 μmol / L) were added to the PC group. After 24 hours of culture, the CCK-8 reagent was operated according to the instructions.

[0071] Positive group cell viability (%): [OD 450 (PC)-OD 450 (BC) / OD 450 (NC)-OD 450 (BC)]×100%OD 450 (PC) is the absorbance of the positive substance treated cell group at 450nm, OD 450 (BC) is the absorbance of the complete culture medium group at 450 nm, OD 450 (NC) is the absorbance of the cell group treated with complete medium at 450 nm.

[0072] 2.2 Effect of positive substance treatment on TRPV1 expression in PC12 cells: ELISA method was used to detect the effect of positive substances on TRPV1 expression in PC12 cells.

[0073] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of 10 cells / mL. The groups were set as NC (control group), CA (capsaicin group), and PC (capsaicin + positive substance group). After 24 hours of culture, 2 mL of complete culture was added to the NC group, 2 mL of 60 μmol / L capsaicin solution was added to the CA group, and 2 mL of 60 μmol / L capsaicin and 1 μmol / L positive substance solution were added to the PC group. After 24 hours of continuous culture, PBS was added to wash once, 200 μL of PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments to destroy the cell membrane, the cell lysate was centrifuged at 2-8°C and 5000×g for 5 minutes to obtain the supernatant. The supernatant was used for subsequent ELISA and BCA experiments. The expression of TRPV1 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0074] 2.3 Effect of positive substance treatment on the expression of substance P in PC12 cells: ELISA was used to detect the effect of capsaicin on the expression of neuropeptide substance P in PC12 cells.

[0075] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC (control group), CA (capsaicin group), and PC (capsaicin + positive substance group). After 24 hours of culture, 2 mL of complete culture was added to the NC group, 2 mL of 60 μmol / L capsaicin solution was added to the CA group, and 2 mL of 60 μmol / L capsaicin and 1 μmol / L positive substance solution were added to the PC group. After 24 hours of continuous culture, PBS was added to wash once, 200 μL of PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments to destroy the cell membrane, the cell lysate was centrifuged at 2-8°C and 5000×g for 5 minutes to obtain the supernatant. The supernatant was used for subsequent ELISA and BCA experiments. The expression of substance P was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0076] 2.4 Effect of positive substance treatment on IL-33 expression in PC12 cells: ELISA was used to detect the effect of capsaicin on the expression of neuroinflammation-related IL-33 in PC12 cells.

[0077] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of 10 cells / mL. The groups were set as NC (control group), CA (capsaicin group), and PC (capsaicin + positive substance group). After 24 hours of culture, 2 mL of complete culture was added to the NC group, 2 mL of 60 μmol / L capsaicin solution was added to the CA group, and 2 mL of 60 μmol / L capsaicin and 1 μmol / L positive substance solution were added to the PC group. After 24 hours of continued culture, PBS was added to wash once, 200 μL of PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments to destroy the cell membrane, the cell lysate was centrifuged at 2-8°C and 5000×g for 5 minutes to obtain the supernatant. The supernatant was used for subsequent ELISA and BCA experiments. The expression of IL-33 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0078] 3. Results Analysis

[0079] All data were processed by GraphPad Prism 9.5.0, and each experiment was repeated at least 3 times. The results were expressed as mean ± standard deviation (Mean ± SD), and the comparison among groups was analyzed by One-way ANOVA. p < 0.05 is indicated by "*"; p < 0.01 is indicated by "**"; p < 0.001 is indicated by "***"; p < 0.0001 is indicated by "****".

[0080] 3.1 Effects of different concentrations of capsaicin on the activity of PC12 cells after 24 h of treatment

[0081] from Figure 1 It can be seen that when untreated, the viability of PC12 cells was 100%, and the cell culture medium was different concentrations of capsaicin solution (20, 40, 60, 80, 100, 120, 140, 160 μmol / L), and the viability of PC12 cells decreased in a dose-dependent manner. The final stimulation concentration was subsequently determined based on the response of the indicators after stimulation.

[0082] 3.2 Effects of capsaicin treatment on TRPV1 channel activation in PC12 cells

[0083] Untreated cells were NC, and the culture medium was capsaicin solution of different concentrations (20, 40, 60, 80, 100 μmol / L). Figure 2 It can be seen that compared with the NC group, 60 μmol / L capsaicin treatment can activate TRPV1 channels, cause calcium ion influx, and increase the intracellular calcium ion fluorescence intensity.

[0084] 3.3 Effects of different concentrations of capsaicin on TRPV1 expression in PC12 cells after 24 h of treatment

[0085] There is increasing evidence that transient receptor potential vanilloid subtype 1 (TRPV1) expression is elevated in patients with sensitive skin and is associated with the severity of sensitive skin symptoms. Figure 3 It can be seen that as the concentration of capsaicin increases, the expression of TRPV1 in PC12 cells first increases and then decreases. The TRPV1 / total protein concentration in the NC group was (271.0±56.44) pg / mg; after 24 hours of injury by adding 60μmol / L capsaicin to the culture medium, the TRPV1 / total protein concentration in the cells increased to (448.5±55.10) pg / mg, P<0.01. The results show that for the TRPV1 index, the stimulation group was successfully stimulated.

[0086] 3.4 Effects of different concentrations of capsaicin on the expression of substance P in PC12 cells after 24 h of treatment

[0087] Substance P plays an important role in the communication between epidermal cells and sensory neurons in the skin. Activated sensory neurons induce the secretion of these neuropeptides. Figure 4 Overall, with the increase of capsaicin concentration, the expression of substance P in PC12 cells first increased and then decreased. The concentration of substance P / total protein in the NC group was (42.83±1.440) pg / mg; after 24 hours of injury by adding 60μmol / L capsaicin to the culture medium, the concentration of substance P / total protein in the cells increased to (48.75±3.138) pg / mg, and reached the highest value after treatment with 80μmol / L, and the concentration of substance P / total protein in the cells increased to (52.84±2.379) pg / mg, P<0.01. The results showed that for the substance P index, the stimulation group was successfully stimulated.

[0088] 3.5 Effects of different concentrations of capsaicin on IL-33 expression in PC12 cells after 24 h of treatment

[0089] IL-33 induces IL-31, which is the main cytokine associated with itch. IL-31 induces the secretion of neuropeptides, thereby inducing neurogenic inflammation and activating nearby nerve branches through axon reflex mechanisms. Figure 5 Overall, as the concentration of capsaicin increased, the expression of IL-33 in PC12 cells first increased and then decreased. The IL-33 / total protein concentration in the NC group was (68.87±4.926) pg / mg; after 24 hours of injury by adding 60μmol / L capsaicin to the culture medium, the IL-33 / total protein concentration in the cells increased to (81.72±5.817) pg / mg, P<0.01. The results showed that for the IL-33 index, the stimulation group was successfully stimulated.

[0090] Taking into account the effects of capsaicin on PC12 cell viability, TRPV1 channel activation, TRPV1 expression, substance P expression, IL-33 expression and other indicators, 60 μmol / L capsaicin was finally selected as the stimulation group.

[0091] 3.6 Effects of different concentrations of positive substances on PC12 cell viability after 24 h of treatment

[0092] from Figure 6It can be seen that when untreated, the viability of PC12 cells was 100%, and when different concentrations of positive substance solutions (0.5, 1, 2, 3, 4, 5, 6, 7 μmol / L) were added to the culture medium, the viability of PC12 cells decreased in a dose-dependent manner. Compared with the NC group, it was considered that the corresponding concentration of cell viability ≥ 90% had no obvious cytotoxicity. After comprehensive consideration, 1 μmol / L positive substance was selected as the subsequent protective substance.

[0093] 3.7 Effects of positive substances on the activation of TRPV1 channels in PC12 cells after 24 h of treatment

[0094] from Figure 7 It can be seen that compared with the NC group, 60 μmol / L capsaicin treatment in the CA group can activate TRPV1 channels, cause calcium ion influx, and increase the fluorescence intensity of intracellular calcium ions; compared with the CA group, the fluorescence intensity of the PC group decreased, indicating that the positive substance can inhibit the activation of TRPV1 channels.

[0095] 3.8 Effect of positive substance treatment on TRPV1 expression in model group PC12 cells after 24 h

[0096] from Figure 8 It can be seen that compared with the NC group, the concentration of TRPV1 / total protein in the cells of the CA group increased; compared with the CA group, the concentration of TRPV1 / total protein in the cells of the PC group decreased significantly (P<0.01), even lower than that of the NC group, indicating that the positive substance has a protective effect against CA stimulation.

[0097] 3.9 Effect of positive substance treatment on the expression of substance P in PC12 cells in the model group after 24 hours

[0098] from Fig. 9 It can be seen that compared with the NC group, the concentration of substance P / total protein in the cells of the CA group was significantly increased, p<0.05; compared with the CA group, the concentration of substance P / total protein in the cells of the PC group was significantly decreased, P<0.05, and even lower than that of the NC group. This shows that positive substances have a protective effect against CA stimulation.

[0099] 3.10 Effect of positive substance treatment on IL-33 expression in model group PC12 cells after 24 h

[0100] from Fig.10 It can be seen that compared with the NC group, the IL-33 / total protein concentration in the CA group cells was significantly increased, p<0.0001; compared with the CA group, the IL-33 / total protein concentration in the PC group cells was significantly decreased, P<0.0001. This shows that the positive substance has a protective effect against CA stimulation.

[0101] Application Examples

[0102] Experimental Materials

[0103] 1. Experimental Materials and Instruments

[0104] 1.1 PC-12 Highly Differentiated Cells

[0105] PC-12 (highly differentiated) was provided by the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0106] 1.2 Main Reagents

[0107] Fetal bovine serum (FBS) was purchased from Vicente Biotechnology (Nanjing) Co., Ltd., CCK-8 reagent was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., BCA kit was purchased from Beijing Bio-Tech Biotechnology Co., Ltd., capsaicin was purchased from MCE, and rat TRPV1 ELISA kit was purchased from Wuhan Fine Biotechnology Co., Ltd.

[0108] 1.3 Instruments

[0109] Cell culture incubator, Thermo; desktop high-speed centrifuge, Eppendorf; desktop high-speed refrigerated centrifuge, Eppendorf; multifunctional microplate reader, TECAN; clean bench, Beijing Shian Kelin Purification Technology Co., Ltd.

[0110] 1.4 Samples to be tested

[0111] The samples are all cosmetic raw materials and are included in the list of used raw materials, namely Sample 1, Sample 2, Sample 3, and Sample 4.

[0112] Samples 1, 2, 3, and 4 will be used below, with cytotoxicity and TRPV1 expression as examples. Other related indicators are not listed one by one, but are also within the scope of protection.

[0113] Application example 1: Using the PC12 cell damage model to evaluate the soothing efficacy of the cosmetic raw material “Sample 1”

[0114] 1. Effect of sample 1 treatment on the activity of highly differentiated PC12 cells: The CCK-8 method was used to detect the cell viability of the control group and different concentrations of sample 1.

[0115] Specific operation: Take PC12 cells with good growth status and add 1×10 5 The cells were inoculated in a 96-well plate at a density of 100 μL cells / mL. The groups were set as BC (blank group), NC (control group), and 1 (sample 1 group), with 6 parallels in each group. After 24 hours of culture, 100 μL of complete culture solution was added to the BC and NC groups, and different concentrations of sample 1 solution (0.25, 0.5, 1, and 2 mg / mL) were added to the 1 group. After another 24 hours of culture, the CCK-8 reagent was operated according to the instructions.

[0116] Cell viability of sample 1 (%): [OD 450(Sample 1)-OD 450 (BC) / OD 450 (NC)-OD 450 (BC)]×100%

[0117] OD 450 (Sample 1) is the absorbance of the sample 1 treated cell group at 450nm, OD 450 (BC) is the absorbance of the complete culture medium group at 450 nm, OD 450 (NC) is the absorbance of the cell group treated with complete medium at 450 nm.

[0118] 2. Effect of sample 1 on the expression of TRPV1 in PC12 cells: ELISA was used to detect the effect of sample 1 on the expression of TRPV1 in PC12 cells.

[0119] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of 10 cells / mL. The groups were set as NC (control group), CA (capsaicin group), and 1 (capsaicin + sample 1 group). After 24 hours of culture, 2 mL of complete culture was added to the NC group, 2 mL of 60 μmol / L capsaicin solution was added to the CA group, and 2 mL of 60 μmol / L capsaicin, 0.25 mg / mL sample 1 solution was added to the 1 group. After 24 hours of continued culture, PBS was added to wash once, 200 μL of cell lysis buffer was added, and lysis was carried out on ice for 30 min-1 h. The cell lysis supernatant was used for subsequent ELISA and BCA experiments. The expression of TRPV1 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0120] from Fig.11 It can be seen that when untreated, the viability of PC12 cells was 100%, and the viability of PC12 cells decreased in a dose-dependent manner when the culture medium was different concentrations of sample 1 solution (0.25, 0.5, 1, 2 mg / mL). Compared with the NC group, it was considered that the corresponding concentration with a cell viability of ≥90% had no obvious cytotoxicity. After comprehensive consideration, the 0.25 mg / mL group was selected as sample 1 group for subsequent evaluation.

[0121] from Fig.12 It can be seen that compared with the NC group, the TRPV1 / total protein concentration in the cells of the CA group was increased; compared with the CA group, the TRPV1 / total protein concentration in the cells of sample group 1 was significantly decreased, P < 0.01, and even lower than that of the NC group.

[0122] Application Example 2: Using the PC12 cell damage model to evaluate the soothing efficacy of the cosmetic raw material “Sample 2”

[0123] 1. Effect of sample 2 treatment on the activity of highly differentiated PC12 cells: The CCK-8 method was used to detect the cell viability of the control group and different concentrations of sample 2 groups.

[0124] Specific operation: Take PC12 cells with good growth status and add 1×10 5 The cells were inoculated in a 96-well plate at a density of 10 cells / mL. The groups were set as BC (blank group), NC (control group), and 2 (sample 2 group), with 6 parallels in each group. After 24 hours of culture, 100 μL of sample 2 solution (5, 10, 20, 40 μmol / L) was added to the BC and NC groups, and different concentrations of sample 2 solution (5, 10, 20, 40 μmol / L) were added to the 2 groups. After another 24 hours of culture, the CCK-8 reagent was operated according to the instructions.

[0125] Cell viability of sample 2 (%): [OD 450 (Sample 2)-OD 450 (BC) / OD 450 (NC)-OD 450 (BC)]×100%

[0126] OD 450 (Sample 2) is the absorbance of the sample 2 treated cell group at 450nm, OD 450 (BC) is the absorbance of the complete culture medium group at 450 nm, OD 450 (NC) is the absorbance of the cell group treated with complete medium at 450 nm.

[0127] 2. Effect of sample 2 on TRPV1 expression in PC12 cells: ELISA was used to detect the effect of sample 2 on TRPV1 expression in PC12 cells.

[0128] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of 10 cells / mL. The groups were set as NC (control group), CA (capsaicin group), and 2 (capsaicin + sample 2 group). After 24 hours of culture, 2 mL of complete culture was added to the NC group, 2 mL of 60 μmol / L capsaicin solution was added to the CA group, and 2 mL of 60 μmol / L capsaicin and 40 μmol / L sample 2 solution was added to the 2 groups. After 24 hours of continued culture, PBS was added to wash once, 200 μL of cell lysis buffer was added, and lysis was carried out on ice for 30 min-1 h. The cell lysis supernatant was used for subsequent ELISA and BCA experiments. The expression of TRPV1 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0129] from Fig.13It can be seen that when untreated, the viability of PC12 cells was 100%, and the culture medium was different concentrations of sample 2 solution (5, 10, 20, 40 μmol / L), and the viability of PC12 cells did not change significantly with the increase of concentration. Compared with the NC group, it is believed that the corresponding concentration with a cell viability of ≥90% has no obvious cytotoxicity. After comprehensive consideration, the 40 μmol / L group was selected as the sample 2 group for subsequent evaluation.

[0130] from Fig.14 It can be seen that compared with the NC group, the TRPV1 / total protein concentration in the cells of the CA group was significantly increased, P < 0.05; compared with the CA group, the TRPV1 / total protein concentration in the cells of sample 2 group was significantly decreased, P < 0.01, and even lower than that of the NC group.

[0131] Application example three: Using the PC12 cell damage model to evaluate the soothing efficacy of the cosmetic raw material “Sample 3”

[0132] 1. Effect of sample 3 treatment on the activity of highly differentiated PC12 cells: The CCK-8 method was used to detect the cell viability of the control group and sample 3 at different concentrations.

[0133] Specific operation: Take PC12 cells with good growth status and add 1×10 5 The cells were inoculated in a 96-well plate at a density of 100 μL cells / mL. The groups were set as BC (blank group), NC (control group), and 3 (sample 3 group), with 6 parallels in each group. After 24 hours of culture, 100 μL of sample 3 solution (0.125, 0.25, 0.375, and 0.5 mmol / L) was added to the BC and NC groups, and different concentrations of sample 3 solution (0.125, 0.25, 0.375, and 0.5 mmol / L) were added to the 3 groups. After another 24 hours of culture, the CCK-8 reagent was used according to its instructions.

[0134] Cell viability of sample 3 groups (%): [OD 450 (Sample 3)-OD 450 (BC) / OD 450 (NC)-OD 450 (BC)]×100%OD 450 (Sample 3) is the absorbance of the sample 3 treated cell group at 450nm, OD 450 (BC) is the absorbance of the complete culture medium group at 450 nm, OD 450 (NC) is the absorbance of the cell group treated with complete medium at 450 nm.

[0135] 2. Effect of sample 3 on TRPV1 expression in PC12 cells: ELISA was used to detect the effect of sample 3 on TRPV1 expression in PC12 cells.

[0136] Specific operation: PC12 cells with good growth status were taken at 1.5×105 The cells were inoculated in a 6-well plate at a density of 10 cells / mL. The groups were set as NC (control group), CA (capsaicin group), and 3 (capsaicin + sample 3 group). After 24 hours of culture, 2 mL of complete culture was added to the NC group, 2 mL of 60 μmol / L capsaicin solution was added to the CA group, and 2 mL of 60 μmol / L capsaicin and 0.25 mmol / L sample 3 solution was added to the 3 group. After 24 hours of continued culture, PBS was added to wash once, 200 μL of cell lysis buffer was added, and lysis was carried out on ice for 30 min-1 h. The cell lysis supernatant was used for subsequent ELISA and BCA experiments. The expression of TRPV1 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0137] from Fig.15 It can be seen that when untreated, the viability of PC12 cells was 100%, and the culture medium was different concentrations of sample 3 solution (0.125, 0.25, 0.375, 0.5mmol / L). The viability of PC12 cells was significantly reduced at 0.5mmol / L, P < 0.01. Compared with the NC group, it is believed that the corresponding concentration of cell viability ≥ 90% has no obvious cytotoxicity. After comprehensive consideration, the 0.25mmol / L group was selected as the sample 3 group for subsequent evaluation.

[0138] from Fig.16 It can be seen that compared with the NC group, the TRPV1 / total protein concentration in the CA group cells was significantly increased, P < 0.05; compared with the CA group, the TRPV1 / total protein concentration in the sample 3 group cells was significantly decreased, P < 0.001, and even lower than that in the NC group.

[0139] Application Example 4: Using the PC12 cell damage model to evaluate the soothing efficacy of the cosmetic raw material “Sample 4”

[0140] 1. Effect of sample 4 treatment on the activity of highly differentiated PC12 cells: The CCK-8 method was used to detect the cell viability of the control group and sample 4 at different concentrations.

[0141] Specific operation: Take PC12 cells with good growth status and add 1×10 5 The cells were inoculated in a 96-well plate at a density of 100 μL cells / mL. The groups were set as BC (blank group), NC (control group), and 4 (sample 4), with 6 parallels in each group. After 24 hours of culture, 100 μL of complete culture solution was added to the BC and NC groups, and different concentrations of sample 4 solution (0.25, 0.5, 1, and 2 mg / mL) were added to the 4 groups. After another 24 hours of culture, the CCK-8 reagent was operated according to the instructions.

[0142] Cell viability of sample 4 groups (%): [OD 450 (Sample 4)-OD 450 (BC) / OD 450(NC)-OD 450 (BC)]×100%OD 450 (Sample 4) is the absorbance of the sample 4 treated cell group at 450nm, OD 450 (BC) is the absorbance of the complete culture medium group at 450 nm, OD 450 (NC) is the absorbance of the cell group treated with complete medium at 450 nm.

[0143] 2. Effect of sample 4 on the expression of TRPV1 in PC12 cells: ELISA was used to detect the effect of sample 4 on the expression of TRPV1 in PC12 cells.

[0144] Specific operation: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of 10 cells / mL. The groups were set as NC (control group), CA (capsaicin group), and 4 (capsaicin + sample 4 group). After 24 hours of culture, 2 mL of complete culture was added to the NC group, 2 mL of 60 μmol / L capsaicin solution was added to the CA group, and 2 mL of 60 μmol / L capsaicin, 0.25 mg / mL sample 4 solution was added to the 4 group. After 24 hours of continued culture, PBS was added to wash once, 200 μL of cell lysis buffer was added, and lysis was carried out on ice for 30 min-1 h. The cell lysis supernatant was used for subsequent ELISA and BCA experiments. The expression of TRPV1 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

[0145] from Fig.17 It can be seen that when untreated, the viability of PC12 cells was 100%, and the culture medium was different concentrations of sample 4 solution (0.25, 0.5, 1, 2 mg / mL), and the viability of PC12 cells decreased in a dose-dependent manner. Compared with the NC group, it is believed that the corresponding concentration with a cell viability of ≥90% has no obvious cytotoxicity. After comprehensive consideration, the 0.25 mg / mL group was selected as the sample 4 group for subsequent evaluation.

[0146] from Fig.18 It can be seen that compared with the NC group, the TRPV1 / total protein concentration in the CA group cells increased; compared with the CA group, the TRPV1 / total protein concentration in the sample 4 group cells decreased and was close to the NC group.

[0147] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics, characterized in that: The following steps are involved: S1. Determine the model stimulation conditions: S11. Determine the effect of capsaicin treatment on the activity of PC12 highly differentiated cells: Use CCK-8 method to detect the cell viability of the control group and different concentrations of capsaicin groups; S12. Determine the effect of capsaicin treatment on the activation of TRPV1 channels in PC12 cells: Use a fluorescence microscope to observe the activation of TRPV1 channels in PC12 cells by capsaicin; S13. Determine the effect of capsaicin treatment on the expression of TRPV1 in PC12 cells: ELISA was used to detect the effect of capsaicin on the expression of TRPV1 in PC12 cells; S14. Determine the effect of capsaicin treatment on the expression of substance P in PC12 cells: ELISA was used to detect the effect of capsaicin on the expression of neuropeptide substance P in PC12 cells; S15. Determine the effect of capsaicin treatment on the expression of IL-33 in PC12 cells: Use ELISA to detect the effect of capsaicin on the expression of neuroinflammation-related IL-33 in PC12 cells.

2. The method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to claim 1, characterized in that: Also includes S2, determine the positive control: S21. Determine the effect of positive substance treatment on the activity of PC12 highly differentiated cells: Use CCK-8 method to detect the cell viability of the control group and the positive substance groups with different concentrations; S22. Determine the effect of positive substance treatment on the activation of TRPV1 channels in PC12 cells: Use a fluorescence microscope to observe the activation of TRPV1 channels in PC12 cells by positive substances; S23. Determine the effect of positive substance treatment on the expression of TRPV1 in PC12 cells: ELISA method was used to detect the effect of positive substance treatment on the expression of TRPV1 in PC12 cells; S24. Determine the effect of positive substance treatment on the expression of neuropeptide substance P in PC12 cells: ELISA method is used to detect the effect of positive substance treatment on the expression of neuropeptide substance P in PC12 cells; S25. Determine the effect of positive substance treatment on the expression of IL-33 in PC12 cells: Use ELISA to detect the effect of positive substance treatment on the expression of neuroinflammation-related IL-33 in PC12 cells.

3. The method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to claim 1, characterized in that: The specific steps of S11 are as follows: PC12 cells with good growth status were taken at 1×10 5 cells / mL were inoculated in a 96-well plate; the groups were set as BC blank group, NC control group, and CA capsaicin group. After culturing for 24 hours, 100 μL of capsaicin solution was added to the BC and NC groups, and different concentrations of capsaicin solution, including 20-160 μmol / L, were added to the CA group; after continuing to culture for 24 hours, the CCK-8 reagent was operated according to the instructions.

4. The method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to claim 3, characterized in that: Cell viability in CA capsaicin group (%): [OD 450 (CA)-OD 450 (BC) / OD 450 (NC)-OD 450 (BC)]×100% OD 450 (CA) is the absorbance of the capsaicin-treated cell group at 450 nm, OD 450 (BC) is the absorbance of the complete culture medium group at 450nm, OD 450 (NC) is the absorbance of the cell group treated with complete medium at 450 nm.

5. The method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to claim 1, characterized in that: The specific steps of S12 are as follows: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of 1.5 cells / mL. The groups were set as NC control group and CA capsaicin group. After culturing for 24 hours, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations, including 20-100 μmol / L, was added to the CA group. After culturing for another 24 hours, the cells were washed twice with HBSS solution, and Fluo-4, AM working solution was added to the cells. The cells were cultured at 37°C for 30 minutes, washed twice with HBSS, and cultured at 37°C for 10 minutes. The cells were immediately photographed under a fluorescence microscope.

6. The method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to claim 5, characterized in that: Fluo-4, AM working solution: Dissolve Fluo-4, AM in anhydrous DMSO to prepare a 2 mmol / L stock solution. Dissolve 50 μg Fluo-4, AM in 22.791 μL anhydrous DMSO. Dilute the Fluo-4, AM solution with basal culture medium to prepare a 4 μmol / L Fluo-4, AM working solution.

7. The method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to claim 1, characterized in that: The specific steps of S13 are as follows: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC control group and CA capsaicin group. After culturing for 24 hours, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations, including 20-100 μmol / L, were added to the CA group. After culturing for another 24 hours, the cells were washed once with PBS, 200 μL of cell lysis buffer was added, and the cells were lysed on ice for 30 min-1 h. The expression of TRPV1 was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

8. The method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to claim 1, characterized in that: The specific steps of S14 are as follows: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of 1.5 cells / mL, and the groups were set as NC control group and CA capsaicin group; after culturing for 24 hours, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations, including 20-100 μmol / L, and the culture was continued for 24 hours, then washed once with PBS, 200 μL of PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments twice to destroy the cell membrane, the cell lysate was centrifuged at 2-8°C and 5000×g for 5 minutes to obtain the supernatant, which was used for subsequent ELISA and BCA experiments. The expression of substance P was detected using an ELISA kit, and protein quantification was performed using a BCA protein quantification kit.

9. The method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to claim 1, characterized in that: The specific steps of S15 are as follows: PC12 cells with good growth status were taken at 1.5×10 5 The cells were inoculated in a 6-well plate at a density of cells / mL. The groups were set as NC control group and CA capsaicin group. After culturing for 24 hours, 2 mL of capsaicin solution was added to the NC group and 2 mL of capsaicin solution of different concentrations, including 20-100 μmol / L, were added to the CA group. After culturing for another 24 hours, the cells were washed once with PBS, 200 μL of PBS was added, the cells were scraped off, and placed at -20°C overnight. After repeated freeze-thaw treatments twice to destroy the cell membrane, the cell lysate was centrifuged at 2-8°C and 5000×g for 5 minutes to obtain the supernatant, which was used for subsequent ELISA and BCA experiments. The ELISA kit was used to detect the expression of IL-33, and the BCA protein quantification kit was used for protein quantification.

10. Application of a method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics, using the method for establishing a PC12 cell model for evaluating the soothing effect of cosmetics according to any one of claims 1 to 9, characterized in that: Evaluating the soothing efficacy of cosmetic raw materials.

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