Construction of an evaluation model for redness removal based on cytokine-stimulated angiogenesis in chicken embryos

Abstract

The invention discloses a method for constructing an evaluation model for removing redness based on cytokine-stimulated chicken embryo angiogenesis: determining the safe concentration of the test substance; dividing the cell culture into a model control group, a negative control group, a positive control group and a test substance group, collecting the supernatant after the incubation; detecting cytokines in the supernatant; selecting fertilized chicken embryos in the rapid growth period of blood vessels, dividing them into a test substance group, a negative control group, a positive control group and a model control group, and recording the distribution of chorioallantoic blood vessels; adding the supernatant collected from the corresponding groups to the chorioallantoic membrane, and recording the distribution of chorioallantoic blood vessels after incubation; counting the bifurcation of blood vessels, calculating the angiogenesis rate and the vascular inhibition rate, and evaluating the effect of removing redness of the test substance. The test substance conditions of the invention are low, and cells are used as the experimental system, with high sensitivity; the experimental cycle is short, and a large number of samples can be quickly detected. Using cells and chicken embryos as experimental models is simple to operate, and the results are intuitive, which is an ideal model for predicting the effect of removing redness.

Description

Technical Field

[0001] The invention belongs to efficacy evaluation technology, and in particular relates to a method for constructing an anti-erythema evaluation model based on cytokine-stimulated chicken embryo angiogenesis. Background Art

[0002] Relevant studies have shown that about 50% of the population suffers from skin sensitivity, including skin tightness, tingling, redness, swollen papules, etc. Daily makeup, staying up late, and ultraviolet rays can all cause skin discomfort and decreased tolerance, making the skin sensitive, leading to sensitive skin. Sensitive skin has always been a hot topic in the field of skin care products, but sensitive skin is not a disease in the general sense, and there is no widely used evaluation model.

[0003] About 43% of people with sensitive skin are troubled by redness of the skin, which is the most common skin problem. Redness of the skin is mainly caused by the dilation of capillaries. Capillaries are mainly located in the dermis of the skin. They are thin in diameter and connected to each other in a network. The blood vessel walls are thin, the blood flow is slow, and the permeability is very high. Under external stimuli, such as strong ultraviolet rays, activated free radicals attack the skin and damage the capillary walls; when the temperature changes suddenly, the capillaries respond to changes in external temperature by contracting and expanding, which may cause the dilation of capillaries and lead to redness. In addition, the skin barrier is damaged and the skin is stimulated to produce an inflammatory response, which is an indirect cause of redness of the skin. External stimuli can cause inflammatory reactions in multiple cells of the skin. This reaction is continuous, interactive and cascading, which is also the complexity of the problem of redness of the skin.

[0004] Mast cells are mainly located near blood vessels and sensory nerves, and are also recruited by chemokines to aggregate at inflammatory sites related to atopic reactions and wound healing. Mast cells are important mediators and effectors of allergic reactions and allergic-like reactions. The cells are filled with many specific granules, which contain a large amount of histamine, heparin, various hydrolases and other inflammatory mediators. They increase the permeability of endothelial cells, cause vasodilation, congestion and exudation, and cause skin erythema and edema. After mast cells are activated, they mediate cell degranulation through a series of signal transduction mechanisms, and release inflammatory factors, which are key effector cells that cause immediate allergic reactions. In healthy tissues, the number of mast cells remains unchanged, while in chronic allergic tissues, the number of mast cells increases significantly. Therefore, mast cells are often used to evaluate the in vitro effects of anti-inflammatory drugs. In addition, basophils also have similar effects. Basophils have many of the same characteristics as mast cells, such as abundant basophilic granules in the cytoplasm and expression of IgE receptor FcεRI on the cell surface. After binding with antigens, the cells can be activated and release granules and inflammatory mediators. They are the main effector cells of IgE-mediated inflammation and are also widely used in the screening of anti-allergic drugs. Although a single cell model can predict the inflammatory effect, it cannot directly reflect the redness-removing effect and has certain limitations.

[0005] Currently, the evaluation methods for removing redness or facial red blood streaks are mainly based on clinical trials, which have long test cycles, insufficient detection throughput, and are mainly based on finished product testing. They are unable to detect cosmetic raw materials and are not ideal for raw material screening in the early stages of research and development. Therefore, the existing redness removal evaluation and testing methods cannot meet current market demand. Summary of the invention

[0006] The purpose of the present invention is to provide a method for constructing an evaluation model for removing redness based on cytokine-stimulated chicken embryo angiogenesis, which has the advantages of simple operation, intuitive experimental results, accurate evaluation results for removing redness, short experimental cycle, and can detect cosmetic raw materials.

[0007] The above-mentioned object of the present invention is achieved by the following technical scheme: a method for constructing an evaluation model for removing redness based on cytokine-stimulated chicken embryo angiogenesis, characterized by comprising the following steps:

[0008] S1. Conduct cell culture, perform cytotoxicity test on the test substance, and determine the safe concentration of the test substance;

[0009] S2. Perform cell culture, which is divided into a model control group, a negative control group, a positive control group and a test substance group. The test substance group is a serum-free culture medium with an activator and a test substance at a safe concentration, the model control group is a serum-free culture medium with an activator, the negative control group is a serum-free culture medium without an activator, and the positive control group is a serum-free culture medium with an activator and a positive substance added, to construct a cell degranulation model, wherein the test substance group is first pre-incubated with a test substance at a safe concentration, then the test substance is removed, the activator is used to stimulate and activate the cells, and a test substance at a safe concentration is added for co-incubation; after the incubation of each group, the supernatant is collected respectively;

[0010] S3. Cytokine detection was performed on the supernatant collected from each group to determine whether the cell degranulation model was successfully established:

[0011] If not, proceed to step S1 until the cell degranulation model is successfully constructed;

[0012] If yes, go to step S4;

[0013] S4. Select fertilized chicken embryos in the rapid blood vessel growth period and divide them into a test substance group, a negative control group, a positive control group and a model control group, expose the chorioallantoic membrane of the chicken embryos in each group, take pictures and record the distribution of blood vessels in the chorioallantoic membrane of the chicken embryos in each group;

[0014] S5, adding the supernatant collected by the model control group in step S2 to the chorioallantoic membrane of chicken embryos in the model control group, adding the supernatant collected by the positive control group in step S2 to the chorioallantoic membrane of chicken embryos in the positive control group, adding the supernatant collected by the negative control group in step S2 to the chorioallantoic membrane of chicken embryos in the negative control group, adding the supernatant collected by the test substance group in step S2 to the chorioallantoic membrane of chicken embryos in the test substance group, and adding the supernatant collected by the test substance group in step S2 to the chorioallantoic membrane of chicken embryos in the test substance group, each group continues to incubate, and after the incubation is completed, taking pictures and recording the vascular distribution of the chorioallantoic membrane of each group of chicken embryos;

[0015] S6. According to the blood vessel distribution of the chorioallantoic membrane of the chicken embryo recorded in each group in steps S4 and S5, the blood vessel bifurcation count was performed, the angiogenesis rate of each group was calculated, and the blood vessel inhibition rate was calculated to evaluate the redness-removing effect of the test substance.

[0016] The present invention combines the culture fluid supernatant containing cytokines with the chicken embryo angiogenesis experiment to detect the red-removing effect of the test object, fills the gap that there is no suitable experimental model for screening red-removing drugs at present, and the test object of the present invention has low condition restrictions, and can be a cosmetic product or a cosmetic raw material; the present invention uses cells as an experimental system, has high sensitivity, can detect trace samples, and the present invention has a short experimental cycle and high test throughput, and can quickly detect a large number of samples. In addition, the present invention uses cells and chicken embryos as experimental models, is simple to operate, and has intuitive results. It is an ideal model for predicting the red-removing effect and can be widely used to evaluate the inhibitory effect of the test object on skin redness.

[0017] In the step S3 of the present invention, the detected cytokines are inflammatory factors TNF-α or IL-6. When the inflammatory factor content of the model control group is significantly higher than that of the negative control group, and the inflammatory factor content of the positive control group is significantly lower than that of the model control group, it indicates that the cell degranulation model is successfully constructed. The above-mentioned "significantly higher than" and "significantly lower than" are statistically analyzed by SPSS software to see whether the difference is statistically significant.

[0018] The positive substance of the present invention is ketotifen fumarate or dexamethasone.

[0019] In the step S6 of the present invention, the vascular bifurcation count obtained by recording the vascular distribution of the chorioallantoic membrane of the chicken embryo in step S4 is V before, and the vascular bifurcation count obtained by recording the vascular distribution of the chorioallantoic membrane of the chicken embryo in step S5 is V after, the calculation formula of the angiogenesis rate and the vascular inhibition rate IR is:

[0020] Angiogenesis rate = V after / V before formula (1)

[0021]

[0022] If IR < 0, it indicates that the test substance has no redness-removing effect;

[0023] If 0≤IR<10, it indicates that the test substance has a slight redness-removing effect;

[0024] If IR≥10, it indicates that the test substance has the effect of removing redness.

[0025] In the step S1 of the present invention, the cell activity of the blank control group is taken as 100%, the relative cell activity of each group of cells is calculated, and the test substance concentration with a relative cell activity greater than or equal to 95% is selected, which is the safe concentration of the test substance.

[0026] In the step S1 of the present invention, the cells are inflammatory cells that are stimulated to produce cytokines, and specifically may be keratinocytes, fibroblasts, mast cells, macrophages, eosinophils or basophils, preferably mast cells.

[0027] In the step S2 of the present invention, the cells are co-cultured cells consisting of one or two of keratinocytes, fibroblasts, mast cells, macrophages, eosinophils and basophils.

[0028] In the step S2 of the present invention, the pre-incubation is 1 to 3 hours and the co-incubation is 0.5 to 2 hours.

[0029] In the step S5 of the present invention, 50 to 200 μL of clear solution is dripped into the chorioallantoic membrane of each group of chicken embryos, and the incubation is continued for 48 to 72 hours.

[0030] In the step S4 of the present invention, the chicken embryo is an SPF-grade fertilized chicken embryo with a growth period of 5 to 9 days.

[0031] Compared with the prior art, the present invention has the following significant effects:

[0032] (1) The present invention utilizes the culture supernatant containing cytokines in combination with the chicken embryo angiogenesis experiment to detect the anti-erythema effect of the test substance, filling the gap of the current lack of a suitable experimental model for screening anti-erythema drugs.

[0033] (2) The test substance of the present invention has low restrictions and can be a cosmetic product or a cosmetic raw material.

[0034] ⑶ The present invention uses cells as an experimental system with high sensitivity and can detect trace amounts of samples.

[0035] (4) The present invention has a short experimental cycle and a high test throughput, and can quickly detect a large number of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Figure 1 This is the image of the negative control group at 0 hours in Example 1 of the present invention;

[0038] Figure 2 This is an image of the negative control group of Example 1 of the present invention after adding the test substance and incubating for 48 hours;

[0039] Figure 3 This is the image of the model control group at 0 hours in Example 1 of the present invention;

[0040] Figure 4 This is an image of the model control group in Example 1 of the present invention after adding the test substance and incubating for 48 hours;

[0041] Figure 5 This is the image of the test substance group of Example 1 of the present invention at 0 hours;

[0042] Figure 6 This is an image of the test substance group of Example 1 of the present invention after incubation for 48 hours with the addition of the test substance. DETAILED DESCRIPTION

[0043] The chorioallantoic membrane (CAM) is an extraembryonic membrane that serves as a gas exchange surface, and its respiratory function is provided by an extensive capillary network. The development of the CAM begins on the fourth day of embryonic incubation, and is formed by the chorionic and allantoic epithelium and the vascularized stroma. Undifferentiated blood vessels grow very rapidly before the eighth day, forming a capillary network. Rapid capillary proliferation continues until the 11th day; thereafter, the endothelial cell mitotic index decreases rapidly, and the vascular system completes the final reticular arrangement on the 18th day. The extensive vascularization during the development of the CAM is very similar to the superficial capillary network of human skin, making it an ideal model for evaluating angiogenic and anti-angiogenic molecules. In cosmetic evaluation, the CAM membrane is also widely used in the detection of conjunctival irritation. Conjunctival irritation reaction can be detected by dropping the test substance on the chick embryo chorioallantoic membrane and observing the vascular bleeding reaction as the basis for detection of irritation. However, conjunctival irritation is significantly different from skin sensitivity redness. Skin sensitivity is a continuous process. The capillary vascular network of the skin does not directly contact with external irritants, but causes the reaction of the main cells of the skin through irritants, and is accumulated and formed by indirect effects such as cytokines (inflammatory factors produced by keratinocytes, growth factors produced by fibroblasts), cell contents (histamine or enzymes released by mast cells), etc., and simple dripping of the test substance is not a suitable detection method. Therefore, the present invention uses inflammatory cells that can stimulate the production of cytokines to co-cultivate with the sample for a period of time, and then uses the co-cultivated culture supernatant to drip on the chicken embryo chorioallantoic membrane to observe the generation inhibition of chicken embryo capillaries, so as to evaluate the redness-removing effect of the test substance.

[0044] Example 1

[0045] The present invention is a method for constructing an evaluation model for removing redness based on cytokine-stimulated chicken embryo angiogenesis. This embodiment is based on establishing an evaluation model for removing redness based on basophil culture fluid and chicken embryo angiogenesis.

[0046] S1. Conduct cell culture, perform cytotoxicity test on the test substance, and determine the safe concentration of the test substance;

[0047] This step is a cytotoxicity test, and the cells used can be inflammatory cells that stimulate the production of cytokines, including keratinocytes, fibroblasts, mast cells, macrophages, eosinophils and basophils, preferably mast cells. The detection method used is the MTT cell activity test method. Water-soluble test substances are prepared using maintenance culture medium, and water-insoluble substances are dissolved using DMSO and diluted 1000 times and added to the culture medium.

[0048] This step specifically includes: preparing a density of 1.0×10 5 / mL of RBL 2H3 cell suspension, inoculate the cell suspension in a 96-well cell culture plate, 100μL per well, and culture for 18h. Dexamethasone (test substance) was prepared into a 10% stock solution using serum-free DMEM culture medium, and then diluted into a series of test concentrations. 100μL of culture solution containing different concentrations of the test substance was added to each well and exposed for 24 hours. Take out the cell culture plate, wash it once with PBS, add 100μL of MTT solution to each well, and incubate in the incubator for 3 hours. Remove the liquid in the well, add 100μLDMSO to each well, place it on an oscillator for 10-15 minutes, measure the absorbance at a wavelength of 570nm on an enzyme reader, and calculate the cell activity. Determine the subsequent test concentration as 20μg / mL (the safe concentration of the test substance).

[0049] S2. Perform cell culture, which is divided into a model control group, a negative control group, a positive control group and a test substance group. The test substance group is a serum-free culture medium with an activator and a test substance at a safe concentration, the model control group is a serum-free culture medium with an activator, the negative control group is a serum-free culture medium without an activator, and the positive control group is a serum-free culture medium with an activator and a positive substance added, to construct a cell degranulation model, wherein the test substance group is first pre-incubated with a test substance at a safe concentration, then the test substance is removed, the activator is used to stimulate and activate the cells, and a test substance at a safe concentration is added for co-incubation; after the incubation of each group, the supernatant is collected respectively;

[0050] This step is for the preparation of inflammatory factors. The cells used can be co-cultured cells composed of one or two of the cells described in step S1. Preferably, a 24-well plate is used for culture, and the cell density is 0.8-1.0×10 5 pcs / mL, 500 μL per well, culture conditions were 37±0.5℃, CO 2 Incubator with 5% content. Preferably, the test substance is prepared in serum-free medium. Preferably, the pre-incubation time is 2 hours and the post-incubation time is 1 hour. Preferably, the concentration of C48 / 80 used in the model construction is 10 μg / mL, and the construction conditions are as shown in the experimental grouping in the following table:

[0051] Negative control group Serum-free medium without C48 / 80 Model control group Serum-free medium containing C48 / 80 Positive control group Serum-free medium containing C48 / 80 and positive substances <![CDATA[ Test group ]]> Serum-free medium containing C48 / 80 and test substances

[0052] (Table 1)

[0053] Among them, the positive substance can be ketotifen fumarate or dexamethasone.

[0054] This step specifically includes: preparing a density of 8×10 5 / mL RBL 2H3 cell suspension, inoculate the cell suspension into 24-well cell culture plates, 500μL per well, and culture for 18 hours. Discard the original culture medium, wash the cells twice with 0.5mL HBSS, add serum-free culture medium containing 20μg / mL dexamethasone to the test group (i.e., the positive control group), add 0.5mL per well, and incubate for 2 hours before placing in the incubator.

[0055] Discard the original culture medium and wash the cells twice with 0.5 mL HBSS. Add according to Table 2, incubate in an incubator for 1 hour, put in an ice bath immediately after the incubation, collect the supernatant in an EP tube under ice-cold conditions, centrifuge at 3000 rpm for 10 minutes, collect the supernatant, and store at -80°C.

[0056] Group Culture conditions Negative control group 1 mL serum-free DMEM Model control group 0.5mL serum-free DMEM + 0.5mL 20ug / mL C48 / 80 <![CDATA[ Test group ]]> 0.5mL 40μg / mL dexamethasone + 0.5mL 20ug / mL C48 / 80

[0057] (Table 2)

[0058] S3. Cytokine detection was performed on the supernatant collected from each group to determine whether the cell degranulation model was successfully established:

[0059] If not, proceed to step S1 until the cell degranulation model is successfully constructed;

[0060] If yes, go to step S4;

[0061] The inflammatory factor detected can be TNF-α or IL-6. When the inflammatory factor content of the model control group is significantly higher than that of the negative control group, and the inflammatory factor content of the positive control group is significantly lower than that of the model control group, it indicates that the experimental model is successfully constructed.

[0062] This step is specifically: using an ELISA kit to determine the content of TNF-a in the supernatant. As shown in Table 3, the TNF-α content of different groups, the model control group compared with the negative control group, the relative content of TNF-α was significantly increased (p < 0.05); compared with the model control group, the relative content of TNF-α in the test group was significantly reduced (p < 0.05), indicating that the model was successfully established and the difference was statistically significant (p < 0.05).

[0063]

[0064] (Table 3)

[0065] S4. Select fertilized chicken embryos in the rapid blood vessel growth period and divide them into a test substance group, a negative control group and a model control group, expose the chorioallantoic membrane of the chicken embryos in each group, take pictures and record the blood vessel distribution of the chorioallantoic membrane of the chicken embryos in each group;

[0066] This step is a chicken embryo angiogenesis experiment, and the chicken embryo is an SPF-grade fertilized chicken embryo, preferably a 5-9-day-old chicken embryo, and more preferably an 8-day-old chicken embryo. Preferably, the incubation conditions are a temperature of 37.5±0.5°C and a humidity of 55-70%, and more preferably the incubation temperature is 37.5°C and the humidity is 60%.

[0067] This step specifically includes: selecting 8-day-old fertilized chicken embryos for grouping into a negative control group, a model control group and a test substance group, with 6 chicken embryos in each group. Use a marker to draw the location of the air chamber, and use tweezers to peel the eggshell along the air chamber to expose the chorioallantoic membrane, and use a stereo microscope to take pictures and record the distribution of blood vessels before adding the test substance.

[0068] S5, adding the supernatant collected by the model control group in step S2 to the chorioallantoic membrane of chicken embryos in the model control group, adding the supernatant collected by the negative control group in step S2 to the chorioallantoic membrane of chicken embryos in the negative control group, adding the supernatant collected by the test substance group in step S2 to the chorioallantoic membrane of chicken embryos in the test substance group, and continuing to incubate each group. After the incubation is completed, taking pictures and recording the vascular distribution of the chorioallantoic membrane of each group of chicken embryos;

[0069] After the photography was completed, 100 μL of culture supernatant was added to each group and the cells were incubated in the incubator for 48 hours. After 48 hours, the cells were photographed using a stereo microscope and the vascular distribution before the addition of the test substance was recorded.

[0070] S6. According to the blood vessel distribution of the chorioallantoic membrane of the chicken embryo recorded in each group in steps S4 and S5, the blood vessel bifurcation count was performed, the angiogenesis rate of each group was calculated, and the blood vessel inhibition rate was calculated to evaluate the redness-removing effect of the test substance.

[0071] The number of vascular bifurcations counted is the number of vascular branch points observed continuously in the same 2-3 matrix fields of view. Preferably, the changes before and after the experiment are observed and compared with the control group, and 2-3 blood vessels with complete vascular branches are counted.

[0072] The counted vascular bifurcations can be observed by coloring with India ink.

[0073] Specifically, the before and after images were compared, and the vascular bifurcations were counted.

[0074] like Figures 1 to 4 As shown, the vascular bifurcation in the model control group was significantly higher than that in the negative control group. Figures 3 to 6 As shown, the vascular bifurcation in the test group was significantly lower than that in the model control group, indicating that 20 μg / mL dexamethasone has a significant inhibitory effect on chicken embryo angiogenesis.

[0075] Example 2

[0076] This example is based on the evaluation of the redness-removing effect of a certain active substance using basophil culture medium and chicken embryo angiogenesis model.

[0077] (1) Cytotoxicity test

[0078] The density of the preparation is 1.0×10 5 / mL RBL 2H3 cell suspension, the cell suspension was inoculated into a 96-well cell culture plate, 100μL per well, and cultured for 18h. A certain active substance was prepared into a 10% stock solution using serum-free DMEM culture medium, and then diluted into a series of test concentrations. 100μL of culture solution containing different concentrations of the test substance was added to each well and exposed for 24 hours. Take out the cell culture plate, wash it once with PBS, add 100μL MTT solution to each well, and incubate in the incubator for 3 hours. Remove the liquid in the well, add 100μL DMSO to each well, place it on an oscillator for 10-15 minutes, measure the absorbance at a wavelength of 570nm on an enzyme reader, and calculate the cell activity. The relative activity of cells at each concentration of a certain active substance is shown in Table 4, and the subsequent test concentrations are determined to be 1.0%, 0.5%, and 0.25%.

[0079] Group(%) <![CDATA[ Viability %)]]> Control 100.00±7.35 10.0000 23.38±0.53 5.0000 69.35±2.85 2.5000 92.12±2.81 1.2500 97.55±1.26 0.6250 95.32±3.79 0.3125 100.43±5.70 0.1563 96.91±4.55 0.0781 97.85±3.32

[0080] (Table 4)

[0081] (2) Preparation of inflammatory factors

[0082] The preparation density is 8×10 5 / mL RBL 2H3 cell suspension, inoculate the cell suspension into 24-well cell culture plates, 500μL per well, and culture for 18 hours. Discard the original culture medium, wash the cells twice with 0.5mL HBSS, add serum-free culture medium containing 1.0%, 0.5%, and 0.25% of a certain active substance to the experimental groups, add 0.5mL to each well, and incubate for 2 hours before placing in the incubator.

[0083] Discard the original culture medium and wash the cells twice with 0.5 mL HBSS. Add the cells according to the experimental groups in Table 5 and incubate them in an incubator for 1 hour. Immediately place in an ice bath after the incubation, collect the supernatant in an EP tube under ice-cold conditions, centrifuge at 3000 rpm for 10 minutes, collect the supernatant, and store at -80°C.

[0084] Group Culture conditions Negative control group 1 mL serum-free DMEM Model control group 0.5mL serum-free DMEM + 0.5mL 20ug / mL C48 / 80 Positive control group 0.5mL 40μg / mL dexamethasone + 0.5mL 20ug / mL C48 / 80 <![CDATA[ Test group ]]> 0.5mL 2.0%, 1%, 0.5% active substance + 0.5mL 20ug / mL C48 / 80

[0085] (Table 5)

[0086] The ELISA kit was used to determine the content of TNF-a in the supernatant. The results are shown in Table 6. The relative content of TNF-α in different groups was significantly increased in the model control group compared with the negative control group (p < 0.05); the relative content of TNF-α in the positive control group was significantly reduced compared with the model control group (p < 0.05), indicating that the model was successfully established. The relative content of TNF-α in the test substance "a certain active substance" at the test concentrations of 1.0%, 0.50%, and 0.25% was significantly reduced compared with the model control group, and the difference was statistically significant (p < 0.05).

[0087]

[0088] (Table 6)

[0089] (3) Chicken embryo angiogenesis experiment

[0090] 8-day-old fertilized chicken embryos were selected and divided into test substance group, model control group, negative control group and positive control group, with 6 chicken embryos in each group. The air cell position was marked with a marker, and the eggshell was peeled off along the air cell with tweezers to expose the chorioallantoic membrane. A stereo microscope was used to take pictures and record the vascular distribution before adding the test substance.

[0091] After the photo was taken, 100 μL of culture supernatant was added to each group and the cells were incubated in the incubator for 48 hours. After 48 hours, a stereo microscope was used to take photos and record the distribution of blood vessels before the addition of the test substance. The pictures before and after were compared, and three chick embryos with good growth were selected to count the vascular bifurcations.

[0092] The angiogenesis inhibition rates are shown in the following table:

[0093]

[0094] (Table 7)

[0095] The results showed that a certain active substance at a concentration of 1% had a significant inhibitory effect on chicken embryo angiogenesis, with an inhibition rate of 36%; a certain active substance at a concentration of 0.5% and 0.25% had no inhibitory effect on chicken embryo angiogenesis.

[0096] Example 3

[0097] This example is based on mast cell culture medium and chicken embryo angiogenesis model to evaluate the redness-removing effect of a skin care solution.

[0098] (1) Cytotoxicity test

[0099] The density of the preparation is 1.0×10 5 / mL P815 cell suspension, inoculate the cell suspension in a 96-well cell culture plate, 100μL per well, and culture for 18h. Prepare a skin care solution into a 10% stock solution using serum-free DMEM culture medium, then dilute it into a series of test concentrations, add 100μL of culture solution containing different concentrations of the test substance to each well, and expose for 24 hours. Take out the cell culture plate, wash it once with PBS, add 100μL MTT solution to each well, and incubate in the incubator for 3 hours. Remove the liquid in the well, add 100μL DMSO to each well, place it on an oscillator for 10-15 minutes, measure the absorbance at a wavelength of 570nm on an enzyme reader, and calculate the cell activity, and determine the subsequent test concentrations as 1.0%, 0.5%, and 0.25%.

[0100] (2) Preparation of inflammatory factors

[0101] The preparation density is 1×10 5 P815 cell suspension of 100 μg / mL was inoculated into a 24-well cell culture plate, 500 μL per well, and cultured for 18 hours. The original culture medium was discarded, and the cells were washed twice with 0.5 mL HBSS. Serum-free culture medium containing 1.0%, 0.5%, and 0.25% of a certain active substance was added to the experimental groups, 0.5 mL per well, and incubated for 2 hours before being placed in the incubator.

[0102] Discard the original culture medium and wash the cells twice with 0.5 mL HBSS. Add according to the group, where the positive sample is ketotifen fumarate. Incubate in an incubator for 1 hour. Immediately after the incubation, ice bath, collect the supernatant in an EP tube under ice-cold conditions, centrifuge at 3000 rpm for 10 minutes, collect the supernatant, and store at -80°C.

[0103] The ELISA kit was used to determine the content of TNF-a in the supernatant. Compared with the negative control group, the relative content of TNF-α in the model control group was significantly increased (p<0.05); compared with the model control group, the relative content of TNF-α in the positive control group was significantly reduced (p<0.05), indicating that the model was successfully established. The relative content of TNF-α in the test substance "a certain skin care solution" was significantly reduced compared with the model control group at the test concentrations of 1.0%, 0.50%, and 0.25%, and the difference was statistically significant (p<0.05).

[0104] (3) Chicken embryo angiogenesis experiment

[0105] 8-day-old fertilized chicken embryos were selected and divided into test substance group, model control group, negative control group and positive control group, with 6 eggs in each group. The air chamber position was marked with a marker, and the eggshell was peeled off along the air chamber with tweezers to expose the chorioallantoic membrane. A stereo microscope was used to take pictures and record the vascular distribution before adding the test substance.

[0106] After the photography was completed, 150 μL of culture supernatant was added to each group and the cells were incubated in the incubator for 48 hours. After 48 hours, a stereo microscope was used to take pictures and record the distribution of blood vessels before the addition of the test substance. The pictures were compared before and after, and three chicken embryos with good growth were selected to count the vascular bifurcations. Each chicken embryo was counted 3 times and the average value was taken. The results are shown in Table 8.

[0107]

[0108] (Table 8)

[0109] The results showed that the skin care solution at concentrations of 1%, 0.5%, and 0.25% had an inhibitory effect on chick embryo angiogenesis, with inhibition rates of 31%, 21%, and 12%, respectively.

Claims

1. A method for constructing an evaluation model for removing redness based on cytokine-stimulated chicken embryo angiogenesis, characterized in that The following steps are involved: S1. Perform cell culture, perform cytotoxicity test on the test substance, and determine the safe concentration of the test substance; the cells are inflammatory cells; S2. Cell culture is carried out and divided into a model control group, a negative control group, a positive control group and a test substance group. The test substance group is a serum-free culture medium with an activator and a test substance at a safe concentration, the model control group is a serum-free culture medium with an activator, the negative control group is a serum-free culture medium without an activator, and the positive control group is a serum-free culture medium with an activator and a positive substance added, and a cell degranulation model is constructed, wherein the test substance group is first pre-incubated with a test substance at a safe concentration, and then the test substance is removed, the activator is used to stimulate and activate the cells, and a test substance at a safe concentration is added for co-incubation; the supernatant of each group is collected after the incubation is completed; the cells are inflammatory cells; S3. Cytokine detection was performed on the supernatant collected from each group to determine whether the cell degranulation model was successfully established: If not, proceed to step S1 until the cell degranulation model is successfully constructed; If yes, go to step S4; The cytokine is TNF-α or IL-6; S4. Select fertilized chicken embryos in the rapid blood vessel growth period and divide them into a test substance group, a negative control group, a positive control group and a model control group, expose the chorioallantoic membrane of the chicken embryos in each group, take pictures and record the distribution of blood vessels in the chorioallantoic membrane of the chicken embryos in each group; S5, adding the supernatant collected by the model control group in step S2 to the chorioallantoic membrane of chicken embryos in the model control group, adding the supernatant collected by the positive control group in step S2 to the chorioallantoic membrane of chicken embryos in the positive control group, adding the supernatant collected by the negative control group in step S2 to the chorioallantoic membrane of chicken embryos in the negative control group, adding the supernatant collected by the test substance group in step S2 to the chorioallantoic membrane of chicken embryos in the test substance group, and adding the supernatant collected by the test substance group in step S2 to the chorioallantoic membrane of chicken embryos in the test substance group, each group continues to incubate, and after the incubation is completed, taking pictures and recording the vascular distribution of the chorioallantoic membrane of each group of chicken embryos; S6. According to the blood vessel distribution of the chorioallantoic membrane of the chicken embryo recorded in each group in steps S4 and S5, the blood vessel bifurcation count was performed, the angiogenesis rate of each group was calculated, and the blood vessel inhibition rate was calculated to evaluate the redness-removing effect of the test substance.

2. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chicken embryo angiogenesis according to claim 1, characterized in that: In step S3, the detected cytokines are inflammatory factors TNF-α or IL-6. When the inflammatory factor content of the model control group is significantly higher than that of the negative control group, and the inflammatory factor content of the positive control group is significantly lower than that of the model control group, it indicates that the cell degranulation model is successfully constructed.

3. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chicken embryo angiogenesis according to claim 2, characterized in that: The positive substance is ketotifen fumarate or dexamethasone.

4. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chick embryo angiogenesis according to claim 3, characterized in that: In step S6, the vascular bifurcation count obtained by recording the vascular distribution of the chorioallantoic membrane of the chicken embryo in step S4 is V before, and the vascular bifurcation count obtained by recording the vascular distribution of the chorioallantoic membrane of the chicken embryo in step S5 is V after, the calculation formula of the angiogenesis rate and the vascular inhibition rate IR is: Angiogenesis rate = V after / V before formula (1) ×100% Official⑵ If IR < 0, it indicates that the test substance has no redness-removing effect; If 0≤IR<10, it indicates that the test substance has a slight redness-removing effect; If IR≥10, it indicates that the test substance has the effect of removing redness.

5. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chick embryo angiogenesis according to claim 4, characterized in that: In step S1, the cell activity of the blank control group is taken as 100%, the relative cell activity of each group of cells is calculated, and the test substance concentration with a relative cell activity greater than or equal to 95% is selected, which is the safe concentration of the test substance.

6. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chick embryo angiogenesis according to claim 5, characterized in that: In the step S1, the cells are keratinocytes, fibroblasts, mast cells, macrophages, eosinophils or basophils.

7. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chick embryo angiogenesis according to claim 6, characterized in that: In step S2, the cells are co-cultured cells consisting of one or two of keratinocytes, fibroblasts, mast cells, macrophages, eosinophils and basophils.

8. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chick embryo angiogenesis according to claim 7, characterized in that: In step S2, the pre-incubation is 1 to 3 hours and the co-incubation is 0.5 to 2 hours.

9. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chick embryo angiogenesis according to claim 8, characterized in that: In step S5, 50-200 μL of the clear solution is dripped into the chorioallantoic membrane of each group of chicken embryos, and the incubation is continued for 48-72 hours.

10. The method for constructing an evaluation model for removing redness based on cytokine-stimulated chick embryo angiogenesis according to claim 9, characterized in that: In step S4, the chicken embryo is an SPF-grade fertilized chicken embryo with a growth period of 5 to 9 days.

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