Application of autophagy inducer in preparation of anti-skin sensitization medicine

Abstract

The invention relates to application of an autophagy inducer in preparation of a drug for resisting skin sensitization. The autophagy inducer is rapamycin. The concentration of the rapamycin is 0.1 to 1 [mu] M. The invention creatively discovers that rapamycin as an autophagy inducer can significantly inhibit release of skin sensitization related cytokines IL-1beta and TNF-alpha. Meanwhile, it is found that rapamycin activates autophagy of skin keratinocytes, and sensitization positive of sensitization antigen presenting cells THP-1 can be converted into sensitization negative.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-skin sensitization drugs, and in particular to application of an autophagy inducer in the preparation of an anti-skin sensitization drug. Background Art

[0002] At present, the entry threshold of the cosmetics industry is too low, which makes the quality of some products uneven, which easily leads to cosmetic-related skin diseases. Cosmetic skin diseases refer to lesions of the skin and appendages caused by the use of cosmetics in people's daily lives, with symptoms such as erythema, papules, desquamation, dry skin and mucous membranes, pigmentation, itching or tingling. Cosmetic allergic reactions are the most common diseases in dermatology clinics, especially facial skin sensitivity, which makes many beauty lovers distressed. In medicine, allergic reactions are also called allergic reactions, which are excessive reactions of the patient's body to one or more substances.

[0003] Inferior cosmetics contain a lot of toxic substances such as fragrances, mineral pigments, heavy metals, etc., which are very irritating to the skin. At present, in the dermatology clinic, we have encountered more and more cases of skin lesions such as erythema, papules, blisters, pigmentation, etc. caused by improper use of cosmetics. Among them, the most common are allergic contact dermatitis and irritant dermatitis. For the study of cosmetic sensitization, the need to find a safe and effective anti-skin sensitization drug is imminent.

[0004] With the introduction of the Adverse Outcome Pathway (AOP) theory and the development of alternative toxicity testing methods, a number of in vitro methods developed for AOPs based on skin sensitization have been accepted by the Organization for Economic Cooperation and Development as testing guidelines, including Direct Peptide Reaction Assay (DPRA), KeratinoSens, U-SENS and Human cell Line Activation Test (h-CLAT), covering the molecular initiation events, key event one (keratinocyte reaction) and key event two (dendritic cell reaction) of AOPs. However, there are still certain limitations based on two-dimensional cell methods such as U-SENS and h-CLAT. For example, there are the following limitations for keratinocyte reaction as the detection endpoint: ① It is difficult to distinguish keratin sensitization reaction from stimulation reaction, and the cells need to be modified to amplify the characteristic signals of sensitization for easy detection; ② The action concentration is limited. For example, for strong stimuli and weak sensitizers, too low concentrations cannot induce cell sensitization reactions, and too high concentrations will cause cell death; ③ Solubility limitations make it impossible to detect samples that are poorly soluble in water; ④ It is impossible to simulate the interaction between keratinocytes and THP-1 cells. The sensitization reaction AOP is a continuous process. After the keratinocytes are activated, they can produce specific cytokines that act on dendritic cells and activate the activation of dendritic cells. The source of the dendritic cell model is mainly the induced culture of CD34 + Dendritic cells obtained from hematopoietic stem cells and peripheral blood, but this type of cell model has shortcomings: differences between laboratories and individuals, high costs, etc. To overcome these limitations, some types of monocytic cell lines with wide sources and strong vitality (THP-1, U937 and MUTZ-3, etc.) have become hot topics in current research. The h-CLAT method directly contacts the test substance with THP-1 cells to achieve the purpose of detection, which is different from the situation when the human body contacts the test substance. The establishment of an in vitro recombinant skin model and a THP-1 cell co-culture model (hereinafter referred to as the co-culture model) can not only play a skin barrier role, but also may activate more pathways and produce perturbations, and can also simulate the interaction between keratinocytes and dendritic cells.

[0005] Autophagy is a highly conserved degradation and recycling process in eukaryotic cells, which plays an important role in the survival and maintenance of cells through the degradation of organelles, proteins and macromolecules and the recycling of decomposition products. Related studies have shown that in normal cells, autophagy is at a low level to maintain the turnover of proteins and organelles; under various physiological and pathological stress conditions, such as growth factor depletion, starvation, hypoxia and chemical-induced stimulation, the level of autophagy will increase.

[0006] As mentioned above, under the stimulation of external allergens such as cosmetics and drugs, allergic contact dermatitis may occur in the skin and mucous membranes, causing damage to the skin keratinocytes at the contact site, producing inflammatory reactions such as edematous erythema or even papules and large scars. In severe cases, epidermal relaxation or even necrosis may occur. Allergic contact dermatitis is a T-cell-mediated immune response. Cytokines, chemokines and co-stimulatory molecules such as IL-1β and TNF-α play an important role in the pathological process of allergic contact dermatitis, and IL-1β, TNF-α and other cytokines can induce cellular autophagy. At the same time, autophagy is a conservative self-digestion and decomposition metabolic process. Autophagy plays an important role in the survival and maintenance of cells through the degradation of organelles, proteins and macromolecules and the recycling of decomposition products. When cells are damaged, autophagy, as a self-protection mechanism, helps cells maintain the stability of the internal environment by specifically degrading damaged or redundant organelles, pathogens or dead cells. Therefore, when external allergens stimulate skin sensitization, the keratinocytes in the skin epidermis are likely to undergo autophagy.

[0007] However, whether autophagy occurs during skin sensitization, the relationship between autophagy and immune regulation, what anti-sensitization effects autophagy has after it occurs, and whether anti-sensitization measures can be developed based on autophagy are still core issues to be studied in this field. Summary of the invention

[0008] To solve the above technical problems, the present invention provides an application of an autophagy inducer in the preparation of an anti-skin sensitization drug. The autophagy inducer is rapamycin, which has potential clinical value as a specific activator to activate the autophagy pathway and may be developed into a new drug with the efficacy of improving skin sensitization.

[0009] To achieve this object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a use of an autophagy inducer in the preparation of an anti-skin sensitization drug, wherein the autophagy inducer is rapamycin;

[0011] The concentration of rapamycin is 0.1-1 μM. The 0.1-1 μM may be, for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM or 1 μM.

[0012] In the present invention, it is found through experimental detection that rapamycin is an autophagy inducer, which can significantly enhance the cell autophagy effect and the anti-skin sensitization effect. It can be used in the field of scientific research to explore the relationship between autophagy and immune regulation, and to develop more autophagy inducers as anti-skin sensitization drugs. Within the above concentration range, skin sensitization can be significantly reduced.

[0013] Preferably, the judgment criteria for skin sensitization include IL-1β positivity and TNF-α positivity.

[0014] In the present invention, IL-1β and TNF-α were found to be positive, which can be judged as skin sensitization, and rapamycin can significantly reduce the content of IL-1β and TNF-α, thereby significantly enhancing the anti-skin sensitization effect.

[0015] Among them, skin sensitization is allergic contact dermatitis (ACD), which is an immune-mediated skin reaction produced by the body after repeated contact of the skin with a certain substance.

[0016] Preferably, the IL-1β positivity means that the IL-1β concentration is greater than 20 pg / mL, for example, it can be 20.1 pg / mL, 30 pg / mL, 50 pg / mL, 80 pg / mL, 100 pg / mL, 1000 pg / mL, 2000 pg / mL, etc., a concentration greater than 20 pg / mL.

[0017] Preferably, the TNF-α positivity means that the TNF-α concentration is greater than 15 pg / mL, for example, it can be 15.1 pg / mL, 30 pg / mL, 50 pg / mL, 80 pg / mL, 100 pg / mL, 1000 pg / mL, 2000 pg / mL, etc., which are greater than 15 pg / mL.

[0018] In the present invention, it was found that the contents of IL-1β and TNF-α increased significantly after treatment with a sensitizer, specifically, the concentration of IL-1β was greater than 20pg / mL and the concentration of TNF-α was greater than 15pg / mL, that is, under this condition, it was judged to be in a skin sensitization state.

[0019] Preferably, the judgment criteria for skin sensitization also include THP-1 cell positivity.

[0020] Preferably, the criterion for determining whether CD86 is positive is RFI. CD86 ≥150, for example, it can be 150, 200, 300, 500, 1000, 2000, etc., which are values ​​greater than 150.

[0021] Preferably, the criterion for determining whether CD54 is positive is RFI. CD54 ≥200, for example, it can be 200, 300, 500, 1000, 2000, or other values ​​greater than 200.

[0022] The criterion for determining whether THP-1 cells are positive also includes a cell survival rate greater than 50%, for example, a survival rate greater than 50%, such as 50.1%, 55%, 90%, 95% or 100%.

[0023] Preferably, the calculation formula of the RFI is:

[0024]

[0025] Among them, MFI is the mean fluorescence intensity (Mean Fluorescence Intensity).

[0026] Preferably, the skin sensitization factors include: any one or a combination of at least two of: sensitizers, temperature, ultraviolet rays, synthetic fiber fabrics, deodorizing products, petroleum products or oils.

[0027] Preferably, the sensitizer includes any one of cinnamaldehyde, hexylcinnamaldehyde, butylphenylmethylpropionic acid, linalool or benzyl salicylate, or a combination of at least two thereof.

[0028] Preferably, the concentration range of the sensitizer is 3-6 μg / mL, for example, 3 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, 5 μg / mL, 5.5 μg / mL or 6 μg / mL.

[0029] Preferably, the cells acted upon by the autophagy inducer are skin keratinocytes.

[0030] Preferably, the drug is in any pharmaceutically acceptable dosage form.

[0031] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0032] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of a carrier, a diluent, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects:

[0034] 1. The present invention creatively discovered that rapamycin, as an autophagy inducer, can significantly inhibit the release of skin sensitization-related cytokines IL-1β and TNF-α. It is also found that rapamycin activates skin keratinocyte autophagy and can convert the sensitized antigen presenting cell THP-1 from positive to negative.

[0035] 2. Rapamycin can significantly enhance the anti-skin sensitization effect within the skin sensitization range of the judgment criteria defined in the present invention, and can produce the autophagy effect to the maximum extent within the defined rapamycin concentration range, thereby resisting sensitization. It can be used in the field of scientific research to explore the relationship between autophagy and immune regulation, and to develop more autophagy inducers as anti-skin sensitization drugs, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The bar graph shows the effect on THP-1 cell survival rate in the co-culture model.

[0037] Figure 2 These are the immunofluorescence experiment diagrams of skin model tissues, among which, Figure A is the result diagram of the co-culture blank group, Figure B is the result diagram of the single model group given 3 μg / mL cinnamaldehyde, Figure C is the result diagram of the co-culture sensitized group, and Figure D is the result diagram of the sensitized combined group.

[0038] Figure 3 It is a bar graph of the IL-1β and TNF-α release levels in different groups.

[0039] Figure 4 Figure 1 is the flow cytometry graph of CD86 in different groups, where Figure A is the result graph of the co-culture blank group, Figure B is the result graph of the co-culture sensitized group, and Figure C is the result graph of the combined sensitized group.

[0040] Figure 5 Figure 1 is the flow cytometry graph of CD54 in different groups, where Figure A is the result graph of the co-culture blank group, Figure B is the result graph of the co-culture sensitized group, and Figure C is the result graph of the sensitized combined group.

[0041] Figure 6 This is a bar graph showing the effects of different concentrations of rapamycin combined with cinnamaldehyde on the survival rate of THP-1 cells. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0043] Sources of reagents and consumables used in the following examples:

[0044] Human peripheral blood mononuclear cell line (THP-1): purchased from American Type Culture Collection (ATCC, VA, USA);

[0045] Cinnamaldehyde: Sigma;

[0046] EpiskinTM epidermal skin model: L'Oréal

[0047] Fetal bovine serum: Gibco

[0048] Antibiotics: Gibco

[0049] Trypsin: Gibco

[0050] Anti-LC-3pAb: BioMed Biotech Co., Ltd.

[0051] Goat anti-rabbit IgG H&L: Alexa 448, Abcam (Shanghai) Trading Co., Ltd.

[0052] Sodium citrate antigen retrieval solution: Shanghai Bio-Tech Biotechnology Co., Ltd.

[0053] DAPI staining solution: Shanghai Bio-Tech Biotechnology Co., Ltd.

[0054] Fluorescence quencher: Shanghai Bio-Tech Biotechnology Co., Ltd.

[0055] CCK-8 kit: Shanghai Bio-Tech Biotech Co., Ltd.

[0056] Interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kit: Solarbio

[0057] Tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kit: Solarbio

[0058] Example 1

[0059] This example investigates the effect of sensitizer concentration on cell viability

[0060] (1) Construction of co-culture model

[0061] First, an in vitro recombinant skin model and THP-1 cell co-culture model were established. The insert dish (basket) with the skin model was transferred to a 12-well plate and cultured for 24 hours using 1640 medium containing 10% fetal bovine serum. Subsequent co-culture was performed after the activity of the skin model was restored. THP-1 cells in the logarithmic growth phase were inoculated in a 12-well plate at a density of 1×10 6 The skin model was transferred to a 12-well plate with THP-1 cells using tweezers and incubated at 37°C with 5% CO 2 and 95% humidity conditions before exposure to sensitizing substances.

[0062] (2) Cell viability determination

[0063] Cinnamaldehyde is a skin sensitizer, but its high concentration can kill THP-1 cells. The skin model was cultured on the upper layer of a 6-well plate, and THP-1 cells were cultured in the culture medium below the skin model. Cinnamaldehyde at concentrations of 0μg / mL, 3μg / mL, 6μg / mL, 12μg / mL, 25μg / mL, and 30μg / mL was applied to the surface of the skin model, and then rinsed after 15 minutes. The CCK-8 method was used to detect the survival rate of THP-1 cells in the lower layer for 24 hours.

[0064] Specific results such as Figure 1 As shown in the figure, the bar graph of the effect of applying the sensitizer cinnamaldehyde on the survival rate of THP-1 cells in the co-culture model on the skin model in the co-culture model shows that cinnamaldehyde in a certain concentration range (3-6 μg / mL) has no effect on the survival of THP-1 cells, and the cell survival rate is greater than 50% when the cinnamaldehyde concentration is below 6 μg / mL.

[0065] Example 2

[0066] This example conducts a skin model tissue immunofluorescence experiment

[0067] The co-culture blank group (only 10% fetal bovine serum 1640 medium was used to maintain the skin model and the underlying THP-1 without any treatment), the co-culture sensitized group (3μg / mL cinnamaldehyde was given after co-culture), the single skin model group was given 3μg / mL cinnamaldehyde and the sensitized combined group (the co-culture model was given 3μg / mL cinnamaldehyde combined with 1μM rapamycin skin model), the above groups were treated for 15 minutes and rinsed, and then placed in formalin fixative for 24 hours. The fixed skin model tissue was made into 8μm thick frozen sections and stored in a -20℃ environment. On the day of the experiment, the sections were taken out of the -20℃ environment and placed at room temperature for 30 minutes. After thawing, they were placed in a section box and 200mL of sodium citrate antigen repair solution was added. The water bath temperature was set to 85℃ for 30 minutes for antigen repair. This step can re-expose the antigen epitope for antibody binding.

[0068] After antigen repair, place at room temperature for 30 minutes. Place on a shaker and rinse with PB buffer three times, 5 minutes each time. Draw a histochemical circle around the sliced ​​tissue with a histochemical pen, and add 10% bovine serum albumin to the histochemical circle for blocking. Add anti-LC-3pAb, i.e. primary antibody (1:200), and incubate at 4°C for 12 hours. Place on a shaker and rinse with PBS buffer three times, 5 minutes each time.

[0069] Add fluorescently labeled goat anti-rabbit IgG H&L (Alexa 448), i.e., biotin-labeled secondary antibody (1:500), was placed in a wet box and placed in an electric hot air drying oven, set at 37°C, and incubated for 30 minutes. Placed on a shaker, rinsed three times with PBS buffer, 5 minutes each time. DAPI staining solution was added for 15 minutes, and then a fluorescence quencher was added and the slide was sealed. The fluorescence distribution and fluorescence intensity in the skin model were observed under a fluorescence microscope and photographed for analysis.

[0070] like Figure 2 The following is an immunofluorescence experiment of skin model tissue in the co-culture model, which shows the expression of related autophagy proteins in each group after 24 hours of treatment. Figure 2 A in the middle is the co-culture blank group, which is an experimental diagram of LC3 fluorescence in skin model tissue, with no green fluorescence. Figure 2 Middle B is a model group that was given 3 μg / mL cinnamaldehyde alone to examine the LC3 fluorescence experiment in the skin model tissue, and green fluorescence was generated. Figure 2 Middle C is the co-culture sensitized group, which examines the LC3 fluorescence experimental diagram in the skin model tissue, and there is almost no green fluorescence. Figure 2 D in the middle is the sensitization combination group. The co-culture model was given 3μg / mL cinnamaldehyde combined with 1μM rapamycin. The LC3 fluorescence experimental diagram in the skin model tissue was examined, and green fluorescence was produced. LC3 is used to mark the dynamic changes of positioning autophagosomes to autophagic lysosomes. The higher the fluorescence intensity, the more autophagy is produced. The above experiment shows that after the autophagy activator is given, the autophagy in the co-culture skin model increases. From the changes from B to C, it can be seen that in the overall co-culture system, autophagy is reduced after the skin model is exposed to the sensitizer. There may be a negative feedback effect in the overall sensitization process, which inhibits the occurrence of autophagy.

[0071] Example 3

[0072] In this example, the IL-1β and TNF-α contents under different treatments were determined.

[0073] (I) Human interleukin-1β (IL-1β) ELSA kit detection (Beijing Solebow Technology Co., Ltd., catalog number SEKH-0002)

[0074] Kit composition: antibody pre-coated ELISA plate, standard, SR1 standard / sample diluent, concentrated biotinylated antibody (100 times), SR2 biotinylated antibody diluent, concentrated enzyme conjugate, SR3 enzyme conjugate diluent, concentrated washing solution, chromogenic substrate, stop solution.

[0075] Take out the test kit from the refrigerator and place it at room temperature for 30 minutes. Wash the plate three times and spin dry, then add SR1 standard (7 concentration points, see SR1 standard gradient dilution) and test samples (i.e., co-culture blank group, co-culture sensitized group, and sensitized combined group cell culture supernatant).

[0076] Take 100 μl of the above standards and samples to the antibody pre-coated ELISA plate, seal the plate and incubate in a 37°C incubator for 90 minutes. After incubation, tap and wash the plate 4 times. Dilute the concentrated biotinylated antibody (100 times) with SR2 biotinylated antibody diluent to the biotinylated working solution (1 times), that is, take 100 μL of concentrated biotinylated antibody and add 9.9 mL of SR2 biotinylated antibody diluent. Add 100 μL of biotinylated antibody working solution to the reaction well, seal the plate and incubate in a 37°C incubator for 60 minutes. After incubation, tap and wash the plate 4 times. Dilute the concentrated enzyme conjugate (100 times) with SR3 enzyme conjugate diluent to the enzyme conjugate working solution (1 times), that is, take 100 μL of concentrated enzyme conjugate and add 9.9 mL of SR3 enzyme conjugate diluent to dilute. Add 100 μL of enzyme conjugate diluent to the reaction wells, seal the plate and incubate in a 37°C incubator for 30 minutes. After incubation, tap and wash the plate 5 times. Add 100 μL of color substrate to the reaction wells, seal the plate and color at 37°C in the dark for 15 minutes. After adding 50 μL of stop solution, immediately measure the absorbance OD value at the specified wavelength (450 nm) using a microplate reader.

[0077] SR1 standard gradient dilution: add 1mL of SR1 standard / sample diluent to IL-1β standard, let it stand for 15 minutes until it is completely dissolved, then gently mix (concentration is 2000pg / mL), and dilute it 2-fold according to the following concentrations: 1000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.25pg / mL, 15.625pg / mL. 1000pg / mL is used as the highest point concentration of the standard curve, and SR1 standard / sample diluent is used as the zero point of the standard curve (0pg / mL).

[0078] (ii) Human tumor necrosis factor-ɑ (TNF-ɑ) ELSA kit detection (Beijing Solebow Technology Co., Ltd., catalog number SEKH0047)

[0079] Kit composition: antibody pre-coated ELISA plate, standard, SR1 standard / sample diluent, concentrated biotinylated antibody (100 times), SR2 biotinylated antibody diluent, concentrated enzyme conjugate, SR3 enzyme conjugate diluent, concentrated washing solution, chromogenic substrate, stop solution.

[0080] Take out the kit from the refrigerator and place it at room temperature for 30 minutes. At the same time, wash the plate three times and dry it, then add SR1 standard (7 concentration points, see SR1 standard gradient dilution) and test samples (i.e., co-culture blank group, co-culture group sensitization group, sensitization combined group cell culture supernatant). Take 100 μL of the above standards and samples to the antibody pre-coated ELISA plate, seal the plate and incubate in a 37°C incubator for 90 minutes. After incubation, tap and wash the plate 4 times. Dilute the concentrated biotinylated antibody (100 times) with SR2 biotinylated antibody diluent to the biotinylated working solution (1 times), that is, take 100 μL of concentrated biotinylated antibody and add 9.9 mL of SR2 biotinylated antibody diluent. Add 100 μl of biotinylated antibody working solution to the reaction well, seal the plate and incubate in a 37°C incubator for 60 minutes. After incubation, tap and wash the plate 4 times. Dilute the concentrated enzyme conjugate (100 times) with SR3 enzyme conjugate diluent to the enzyme conjugate working solution (1 times), that is, take 100 μL of concentrated enzyme conjugate and add 9.9 mL of SR3 enzyme conjugate diluent to dilute. Add 100 μL of enzyme conjugate diluent to the reaction well, seal the plate and incubate in a 37°C incubator for 30 minutes. After incubation, tap and wash the plate 5 times. Add 100 μL of color substrate to the reaction well, seal the plate and color for 15 minutes at 37°C in the dark. After adding 50 μL of stop solution, immediately measure the absorbance OD value at the specified wavelength (450nm) with an enzyme reader.

[0081] SR1 standard gradient dilution: add 1 mL of SR1 standard / sample diluent to TNF-ɑ standard, let stand for 15 minutes until it is completely dissolved, then gently mix (concentration is 1000 pg / mL), and dilute 2-fold according to the following concentrations: 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.625 pg / mL. 1000 pg / mL is used as the highest point concentration of the standard curve, and SR1 standard / sample diluent is used as the zero point of the standard curve (0 pg / mL).

[0082] The results are as follows Figure 3 As shown, after adding the sensitizer to the co-culture group, it can be found that the IL-1β and TNF-α levels increased significantly, that is, the skin sensitization state was successfully established, that is, the IL-1β and TNF-α levels were significantly different from those of the blank group, which was judged as skin sensitization (p<0.05). When 1μM rapamycin was added, it was found that the IL-1β and TNF-α levels were significantly reduced, indicating that 1μM rapamycin can inhibit skin sensitization. In the process of skin sensitization, there are literature reports that it will be accompanied by the release of some cytokines, and it also involves some complex inflammation and other pathological pathways, but there is no relevant report on autophagy research. The present invention discloses for the first time the role of autophagy inhibitors in skin sensitization.

[0083] Example 4

[0084] This example conducts a human cell line activation test

[0085] Sample treatment: divided into three groups, the first group is the co-culture blank group; the second group is the co-culture sensitization group (3μg / mL cinnamaldehyde was added to the co-culture model); the third group is the sensitization combination group (3μg / mL cinnamaldehyde and 1μM rapamycin were added to the co-culture model). The above groups were treated for 15 minutes and then washed, and the expression of CD54 and CD86 in THP-1 cells was detected 24 hours later.

[0086] The above cells were transferred to 5mL sterile EP tubes and centrifuged at 1000 rpm for 5 minutes in a 4℃ centrifuge. The supernatant was discarded and the cells were washed with 1mL of flow cytometry buffer and centrifuged at 1000 rpm to complete two washes. After washing, 600μL / tube of blocking solution was used for blocking at 4℃ for 15 minutes. After blocking, the cells were divided into 3 parts in 1.5mL EP tubes, each with about 180μL. Centrifuged at 1000 rpm for 3 minutes at 4℃, and the supernatant was discarded. 50μL of FITC-labeled mouse monoclonal antibody CD86 and FITC-labeled mouse monoclonal antibody CD54 were added and incubated at 4℃ for 30 minutes in the dark. After staining, the cells were washed with 200μL / tube of flow cytometry buffer, centrifuged at 1000 rpm for 3 minutes, and washed twice, and then the cells were resuspended with flow cytometry buffer. Before loading, add 50 μL PI dye to each tube, and flow cytometry can be performed after 5 minutes. Select the FL1 channel to detect FITC staining, that is, the expression of CD86 and CD54, and select the FL3 channel to check the PI staining, that is, to detect the cell survival rate. After loading, different methods are established according to the different content of the measurement. It is necessary to obtain scatter plots, cross-shaped two-parameter histograms, and histograms of different gates after the circle gate, calculate the relative fluorescence intensity RFI value, and obtain the cell viability at different drug concentrations based on the FSC / SSC graph and the parameters in the graph.

[0087] THP-1 cells positive including RFI CD86 ≥150 and RFI CD54 ≥200, and the corresponding cell survival rate is greater than 50%. The specific results are shown in Table 1, and the calculation formula is as follows;

[0088]

[0089] Table 1

[0090] / Co-culture blank group Co-culture sensitized group Sensitization combined group <![CDATA[RFI CD86 ]]> 128.13 177.39 139.18 <![CDATA[RFI CD54 ]]> 118.68 332.60 150.76

[0091] Specific results such as Figure 4 and Figure 5 As shown, Figure 4 The flow cytometry graphs of the CD86 index in the co-culture blank group, co-culture sensitized group (3 μg / mL cinnamaldehyde), and sensitized combined group (3 μg / mL cinnamaldehyde + 1 μM rapamycin) are shown in the following table. Figure 4 The RFICD86 of A co-culture blank group was 128.13, 4B co-culture sensitized group (3μg / mL cinnamaldehyde) RFICD86 was 177.39; the RFICD86 of combined sensitization group (3μg / mL cinnamaldehyde + 1μM rapamycin) was 139.18.

[0092] Figure 5 The flow cytometry graphs of the CD54 index in the co-culture blank group, co-culture sensitized group (3 μg / mL cinnamaldehyde), and combined sensitized group (3 μg / mL cinnamaldehyde + 1 μM rapamycin) are shown in the following table. Figure 5 A. RFI of the co-culture blank group CD54 118.68, 5B is the co-culture sensitization group (3μg / mL cinnamaldehyde) RFI CD54 The RFI of the combined sensitization group (3 μg / mL cinnamaldehyde + 1 μM rapamycin) was 332.60. CD54 It is 150.76.

[0093] The above results show that the RFI of the co-culture sensitized group (3 μg / mL cinnamaldehyde) CD86 ≥150 and RFI CD54 ≥200, and the corresponding cell survival rate is greater than 50%, the result is positive for sensitization; combined sensitization group (3μg / mL cinnamaldehyde + 1μM rapamycin) RFI CD86 <150 and RFI CD54 <200, the result is negative for sensitization.

[0094] Example 5

[0095] This example tests the concentration range of rapamycin

[0096] The experimental procedures were the same as those in implementation 4, except that combined sensitization group 1 was 3 μg / mL cinnamaldehyde + 2 μM rapamycin, combined sensitization group 2 was 3 μg / mL cinnamaldehyde + 3 μM rapamycin, and combined sensitization group 3 was 3 μg / mL cinnamaldehyde + 4 μM rapamycin.

[0097] Depend on Figure 6 It can be seen that when rapamycin is 4 μM, the corresponding cell survival rate is less than 50%, and the experiment is not valid. The relative fluorescence intensity RFI value of combined sensitization group 1 and combined sensitization group 2 is calculated:

[0098] Table 2

[0099] / Co-culture blank group Combined sensitization group 1 Combined sensitization group 2 <![CDATA[RFI CD86 ]]> 128.13 132.46 124.57 <![CDATA[RFI CD54 ]]> 118.68 158.65 141.26

[0100] As shown in Table 2, rapamycin concentrations of 1-3 μM can inhibit the positive sensitization caused by the sensitizer cinnamaldehyde.

[0101] In summary, the present invention establishes a co-culture model of an in vitro skin model and antigen presenting cell THP-1, applies different concentrations of sensitizer cinnamaldehyde to the skin model, and finds a suitable concentration of 3μg / mL that does not affect cell survival. The co-culture model is treated with 3μg / mL cinnamaldehyde and 3μg / mL cinnamaldehyde combined with 1μM rapamycin for 15min and then rinsed. After 24 hours, the expression of related autophagy proteins (LC3) in each group is checked. The results show that after the autophagy activator is given, the autophagy production in the skin model increases. Take the co-culture model culture fluid, and perform OD value detection according to the instructions of the Elisa kit; the results show that 3μg / mL cinnamaldehyde causes an increase in the release of IL-1β and TNF-α in the culture fluid, and 3μg / mL cinnamaldehyde combined with 1μM rapamycin reduces the release of IL-1β and TNF-α caused by 3μg / mL cinnamaldehyde in the culture fluid. THP-1 cells in the co-culture model were stained for CD54 and CD86 and flow cytometry was performed; the results showed that the co-culture model administered 3 μg / mL cinnamaldehyde resulted in positive CD54 and CD86 in THP-1 cells; 3 μg / mL cinnamaldehyde combined with 1 μM rapamycin resulted in negative CD54 and CD86 in THP-1 cells. The above data indicate that the autophagy activator rapamycin can inhibit the skin sensitization effect caused by the sensitizer cinnamaldehyde.

[0102] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of an autophagy inducer in the preparation of an anti-skin sensitization drug, characterized in that: The autophagy inducer is rapamycin; The concentration of rapamycin is 0.1-1 μM.

2. The use of the autophagy inducer according to claim 1 in the preparation of an anti-skin sensitization drug, characterized in that: The judgment criteria for skin sensitization include IL-1β positivity and TNF-α positivity.

3. Use of the autophagy inducer according to claim 1 or 2 in the preparation of an anti-skin sensitization drug, characterized in that: The IL-1β positive means that the IL-1β concentration is greater than 20pg / mL; Preferably, the TNF-α positivity means that the TNF-α concentration is greater than 15 pg / mL.

4. Use of the autophagy inducer according to any one of claims 1 to 3 in the preparation of an anti-skin sensitization drug, characterized in that: The judgment criteria for skin sensitization also include THP-1 cell positivity.

5. Use of the autophagy inducer according to any one of claims 1 to 4 in the preparation of an anti-skin sensitization drug, characterized in that: The THP-1 cell positivity includes CD54 positivity and / or CD86 positivity in THP-1 cells; Preferably, the criterion for determining whether CD86 is positive is RFI. CD86 ≥150; Preferably, the criterion for determining whether CD54 is positive is RFI. CD54 ≥200; Preferably, the calculation formula of the RFI is: Among them, MFI is the mean fluorescence intensity.

6. Use of the autophagy inducer according to claim 5 in the preparation of an anti-skin sensitization drug, characterized in that: The skin sensitization factors include: any one of sensitizers, temperature, ultraviolet rays, synthetic fiber fabrics, deodorant products, petroleum products or oils, or a combination of at least two of them.

7. Use of the autophagy inducer according to claim 6 in the preparation of an anti-skin sensitization drug, characterized in that: The sensitizer includes: any one or a combination of at least two of cinnamaldehyde, hexylcinnamaldehyde, butylphenylmethylpropionic acid, linalool or benzyl salicylate; Preferably, the concentration of the sensitizer is in the range of 3-6 μg / mL.

8. Use of the autophagy inducer according to any one of claims 1 to 7 in the preparation of an anti-skin sensitization drug, characterized in that: The cells acted upon by the autophagy inducer are skin keratinocytes.

9. Use of the autophagy inducer according to any one of claims 1 to 8 in the preparation of an anti-skin sensitization drug, characterized in that: The drug is in any pharmaceutically acceptable dosage form; Preferably, the drug further contains pharmaceutically acceptable excipients.

10. Use of the autophagy inducer according to claim 9 in the preparation of an anti-skin sensitization drug, characterized in that: The pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, diluents, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

Images